Cancer stem cells in oesophageal squamous cell carcinoma: Identification, prognostic and treatment perspectives

Cancer stem cells (CSCs) are a vital subpopulation of cells to target for the treatment of cancers. In oesophageal squamous cell carcinoma (ESCC), there are several markers such as CD44, ALDH, Pygo2, MAML1, Twist1, Musashi1, Side population (SP), CD271 and CD90 that have been proposed to identify the cancer stem cells in individual cancer masses. It has also been demonstrated that stem cell markers like ALDH1, HIWI, Oct3/4, ABCG2, SOX2, SALL4, BMI-1, NANOG, CD133 and podoplanin are associated with patient's prognosis, pathological stages, cancer recurrence and therapy resistance. Finding new cancer stem cell targets or designing drugs to manipulate the known molecular targets in CSCs could be useful for improvements in clinical outcomes of the disease. To conclude, data suggest that CSCs in oesophageal squamous cell carcinoma are related to resistance to therapy and poor prognosis of patients with ESCC. Therefore, innovative insights into CSC biology and CSC-targeted therapies will help to achieve more effective management of patients with oesophageal squamous cell carcinoma.

Hematopoietic Stem Cell Development and Transcriptional Regulation

The continuous production of blood cells, a process termed hematopoiesis, is sustained throughout the lifetime of an individual by a relatively small population of cells known as hematopoietic stem cells (HSCs). HSCs are unique cells characterized by their ability to self-renew and give rise to all types of mature blood cells. Given their high proliferative potential, HSCs need to be tightly regulated on the cellular and molecular levels or could otherwise turn malignant. On the other hand, the tight regulatory control of HSC function also translates into difficulties in culturing and expanding HSCs in vitro. In fact, it is currently not possible to maintain or expand HSCs ex vivo without rapid loss of self-renewal. Increased knowledge of the unique features of important HSC niches and of key transcriptional regulatory programs that govern HSC behavior is thus needed. Additional insight in the mechanisms of stem cell formation could enable us to recapitulate the processes of HSC formation and self-renewal/expansion ex vivo with the ultimate goal of creating an unlimited supply of HSCs from e.g. human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS) to be used in therapy. We thus asked: How are hematopoietic stem cells formed and in what cellular niches does this happen (Papers I, II)? What are the molecular mechanisms that govern hematopoietic stem cell development and differentiation (Papers III, IV)? Importantly, we could show that placenta is a major fetal hematopoietic niche that harbors a large number of HSCs during midgestation (Paper I)(Gekas et al., 2005). In order to address whether the HSCs found in placenta were formed there we utilized the Runx1-LacZ knock-in and Ncx1 knockout mouse models (Paper II). Importantly, we could show that HSCs emerge de novo in the placental vasculature in the absence of circulation (Rhodes et al., 2008). Furthermore, we could identify defined microenvironmental niches within the placenta with distinct roles in hematopoiesis: the large vessels of the chorioallantoic mesenchyme serve as sites of HSC generation whereas the placental labyrinth is a niche supporting HSC expansion (Rhodes et al., 2008). Overall, these studies illustrate the importance of distinct milieus in the emergence and subsequent maturation of HSCs. To ensure proper function of HSCs several regulatory mechanisms are in place. The microenvironment in which HSCs reside provides soluble factors and cell-cell interactions. In the cell-nucleus, these cell-extrinsic cues are interpreted in the context of cell-intrinsic developmental programs which are governed by transcription factors. An essential transcription factor for initiation of hematopoiesis is Scl/Tal1 (stem cell leukemia gene/T-cell acute leukemia gene 1). Loss of Scl results in early embryonic death and total lack of all blood cells, yet deactivation of Scl in the adult does not affect HSC function (Mikkola et al., 2003b. In order to define the temporal window of Scl requirement during fetal hematopoietic development, we deactivated Scl in all hematopoietic lineages shortly after hematopoietic specification in the embryo . Interestingly, maturation, expansion and function of fetal HSCs was unaffected, and, as in the adult, red blood cell and platelet differentiation was impaired (Paper III)(Schlaeger et al., 2005). These findings highlight that, once specified, the hematopoietic fate is stable even in the absence of Scl and is maintained through mechanisms that are distinct from those required for the initial fate choice. As the critical downstream targets of Scl remain unknown, we sought to identify and characterize target genes of Scl (Paper IV). We could identify transcription factor Mef2C (myocyte enhancer factor 2 C) as a novel direct target gene of Scl specifically in the megakaryocyte lineage which largely explains the megakaryocyte defect observed in Scl deficient mice. In addition, we observed an Scl-independent requirement of Mef2C in the B-cell compartment, as loss of Mef2C leads to accelerated B-cell aging (Gekas et al. Submitted). Taken together, these studies identify key extracellular microenvironments and intracellular transcriptional regulators that dictate different stages of HSC development, from emergence to lineage choice to aging.

The role of mast cells in ischemic and hemorrhagic brain injury

Stroke, ischemic or hemorrhagic, belongs among the foremost causes of death and disability worldwide. Massive brain swelling is the leading cause of death in large hemispheric strokes and is only modestly alleviated by available treatment. Thrombolysis with tissue plasminogen activator (TPA) is the only approved therapy in acute ischemic stroke, but fear of TPA-mediated hemorrhage is often a reason for withholding this otherwise beneficial treatment. In addition, recanalization of the occluded artery (spontaneously or with thrombolysis) may cause reperfusion injury by promoting brain edema, hemorrhage, and inflammatory cell infiltration. A dominant event underlying these phenomena seems to be disruption of the blood-brain barrier (BBB). In contrast to ischemic stroke, no widely approved clinical therapy exists for intracerebral hemorrhage (ICH), which is associated with poor outcome mainly due to the mass effect of enlarging hematoma and associated brain swelling. Mast cells (MCs) are perivascularly located resident inflammatory cells which contain potent vasoactive, proteolytic, and fibrinolytic substances in their cytoplasmic granules. Experiments from our laboratory showed MC density and their state of granulation to be altered early following focal transient cerebral ischemia, and degranulating MCs were associated with perivascular edema and hemorrhage. (I) Pharmacological MC stabilization led to significantly reduced ischemic brain swelling (40%) and BBB leakage (50%), whereas pharmacological MC degranulation raised these by 90% and 50%, respectively. Pharmacological MC stabilization also revealed a 40% reduction in neutrophil infiltration. Moreover, genetic MC deficiency was associated with an almost 60% reduction in brain swelling, 50% reduction in BBB leakage, and 50% less neutrophil infiltration, compared with controls. (II) TPA induced MC degranulation in vitro. In vivo experiments with post-ischemic TPA administration demonstrated 70- to 100-fold increases in hemorrhage formation (HF) compared with controls HF. HF was significantly reduced by pharmacological MC stabilization at 3 (95%), 6 (75%), and 24 hours (95%) of follow-up. Genetic MC deficiency again supported the role of MCs, leading to 90% reduction in HF at 6 and 24 hours. Pharmacological MC stabilization and genetic MC deficiency were also associated with significant reduction in brain swelling and in neutrophil infiltration. Importantly, these effects translated into a significantly better neurological outcome and lower mortality after 24 hours. (III) Finally, in ICH experiments, pharmacological MC stabilization resulted in significantly less brain swelling, diminished growth in hematoma volume, better neurological scores, and decreased mortality. Pharmacological MC degranulation produced the opposite effects. Genetic MC deficiency revealed a beneficial effect similar to that found with pharmacological MC stabilization. In sum, the role of MCs in these clinically relevant scenarios is supported by a series of experiments performed both in vitro and in vivo. That not only genetic MC deficiency but also drugs targeting MCs could modulate these parameters (translated into better outcome and decreased mortality), suggests a potential therapeutic approach in a number of highly prevalent cerebral insults in which extensive tissue injury is followed by dangerous brain swelling and inflammatory cell infiltration. Furthermore, these experiments could hint at a novel therapy to improve the safety of thrombolytics, and a potential cellular target for those seeking novel forms of treatment for ICH.

Promotion of a cancer-like phenotype, through chronic exposure to inflammatory cytokines and hypoxia in a bronchial epithelial cell line model

Globally, lung cancer accounts for approximately 20% of all cancer related deaths. Five-year survival is poor and rates have remained unchanged for the past four decades. There is an urgent need to identify markers of lung carcinogenesis and new targets for therapy. Given the recent successes of immune modulators in cancer therapy and the improved understanding of immune evasion by tumours, we sought to determine the carcinogenic impact of chronic TNF-α and IL-1β exposure in a normal bronchial epithelial cell line model. Following three months of culture in a chronic inflammatory environment under conditions of normoxia and hypoxia (0.5% oxygen), normal cells developed a number of key genotypic and phenotypic alterations. Important cellular features such as the proliferative, adhesive and invasive capacity of the normal cells were significantly amplified. In addition, gene expression profiles were altered in pathways associated with apoptosis, angiogenesis and invasion. The data generated in this study provides support that TNF-α, IL-1β and hypoxia promotes a neoplastic phenotype in normal bronchial epithelial cells. In turn these mediators may be of benefit for biomarker and/or immune-therapy target studies. This project provides an important inflammatory in vitro model for further immuno-oncology studies in the lung cancer setting.

The Risk of Cancer Associated with Immunosuppressive Therapy for Skin Diseases

The possible carcinogenic risk of immunosuppressive therapies is an important issue in everyday clinical practise. Carcinogenesis is a slow multi step procedure, thus a long latency period is needed before cancer develops. PUVA therapy is used for many skin diseases including psoriasis, early stage cutaneous T cell lymphoma, atopic dermatitis, palmoplantar pustulosis and chronic eczema. There has been concern about the increased melanoma risk associated to PUVA therapy, which has previously been associated with an increased risk on non-melanoma skin cancer, especially squamous cell carcinoma. The increased risk of basal cell carcinoma (BCC) is also documented but it is modest compared to squamous cell carcinoma (SCC). This thesis evaluated melanoma and noncutaneous cancer risk associated to PUVA, and the persistence of nonmelanoma cancer risk after the cessation of PUVA treatment. Also, the influence of photochemotherapy to the development of secondary cancers in cutaneous T cell lymphoma and the role of short term cyclosporine in later cancer development in inflammatory skin diseases were evaluated. The first three studies were performed on psoriasis patients. The risk of melanoma started to increase 15 years after the first treatment with PUVA. The risk was highest among persons who had received over 250 treatments compared to those under 250 treatments. In noncutaneous cancer, the overall risk was not increased (RR=1.08,95% CI=0.93-1.24), but significant increases in risk were found in thyroid cancer, breast cancer and in central nervous system neoplasms. These cancers were not associated to PUVA. The increased risk of SCC was associated to high cumulative UVA exposure in the PUVA regimen. The patients with high risk had no substantial exposure to other carcinogens. In BCC there was a similar but more modest tendency. In the two other studies, the risk of all secondary cancers (SIR) in CTCL patients was 1.4 (95% CI=1.0-1.9). In separate sites, the risk of lung cancer, Hodgkin and non-Hodgkin lymphomas were increased. PUVA seemed not to contribute to any extent to the appearance of these cancers. The carcinogenity of short-term cyclosporine was evaluated in inflammatory skin diseases. No increased risk for any type of cancer including the skin cancers was detected. To conclude, our studies confirm the increased skin cancer risk related to PUVA treatment in psoriasis patients. In clinical practice, this has led to a close and permanent follow-up of patients treated with PUVA. In CTCL patients, PUVA treatment did not contribute to the development of secondary cancers. We could not detect any increase in the risk of cancer in patients treated with short term cyclosporine, unlike in organ transplant patients under such long-term therapy.

Pathological changes at the target tissue level in Sjögren's syndrome and their effect on the exocrine function of the salivary glands

Sjögren s syndrome (SS) is a common autoimmune disease affecting the lacrimal and salivary glands. SS is characterized by a considerable female predominance and a late age of onset, commonly at the time of adreno- and menopause. The levels of the androgen prohormone dehydroepiandrosterone-sulphate (DHEA-S) in the serum are lower in patients with SS than in age- and sex-matched healthy control subjects. The eventual systemic effects of low androgen levels in SS are not currently well understood. Basement membranes (BM) are specialized layers of extracellular matrix and are composed of laminin (LM) and type IV collagen matrix networks. BMs deliver messages to epithelial cells via cellular LM-receptors including integrins (Int) and Lutheran blood group antigen (Lu). The composition of BMs and distribution of LM-receptors in labial salivary glands (LSGs) of normal healthy controls and patients with SS was assessed. LMs have complex and highly regulated distribution in LSGs. LMs seem to have specific tasks in the dynamic regulation of acinar cell function. LM-111 is important for the normal acinar cell differentiation and its expression is diminished in SS. Also LM-211 and -411 seem to have some acinar specific functional tasks in LSGs. LM-311, -332 and -511 seem to have more general structure maintaining and supporting roles in LSGs and are relatively intact also in SS. Ints α3β1, α6β1, α6β4 and Lu seem to supply structural basis for the firm attachment of epithelial cells to the BM in LSGs. The expression of Ints α1β1 and α2β1 differed clearly from other LM-receptors in that they were found almost exclusively around the acini and intercalated duct cells in salivons suggesting some type of acinar cell compartment-specific or dominant function. Expression of these integrins was lower in SS compared to healthy controls suggesting that the LM-111 and -211-to-Int α1β1 and α2β1 interactions are defective in SS and are crucial to the maintenance of the acini in LSGs. DHEA/DHEA-S concentration in serum and locally in saliva of patients with SS seems to have effects on the salivary glands. These effects were first detected using the androgen-dependent CRISP-3 protein, the production and secretion of which were clearly diminished in SS. This might be due to the impaired function of the intracrine DHEA prohormone metabolizing machinery, which fails to successfully convert DHEA into its active metabolites in LSGs. The progenitor epithelial cells from the intercalated ductal area of LSGs migrate to the acinar compartment and then undergo a phenotype change into secretory acinar cells. This migration and phenotype change seem to be regulated by the LM-111-to-Int α1β1/Int α2β1 interactions. Lack of these interactions could be one factor limiting the normal remodelling process. Androgens are effective stimulators of Int α1β1 and α2β1 expression in physiologic concentrations. Addition of DHEA to the culture medium had effective stimulating effect on the Int α1β1 and α2β1 expression and its effect may be deficient in the LSGs of patients with SS.

Auger-emitter radiochemotherapy in squamous cell cancers : in vitro and in vivo experiments with In-111-BLMC

Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer worldwide. Despite advances in combined modality therapy (surgery, radiotherapy, chemotherapy) the 5-year survival rate in stage III and IV disease remains at 40% - 60%. Short-range Auger-electron emitters, such as In-111 and In-114m, tagged with a drug, molecule, peptide, protein or nanoparticles brought in close proximity to nuclear DNA represent a fascinating alternative for treating cancer. In this thesis, we studied the usefulness of Indium-111-bleomycin complex (In-111-BLMC) in the diagnostics and potential therapy of HNSCC using in vitro HNSCC cell lines, in vivo nude mice, and in vivo HNSCC patients. In in vitro experiments with HNSCC cell lines, the sensitivity to external beam radiation, BLM, In-111-BLMC, and In-111-Cl3 was studied using the 96-well plate clonogenic assay. The influence of BLM and In-111-BLMC on the cell cycle was measured with flow cytometry. In in vivo nude mice xenograft studies, the activity ratios of In-111-BLMC were obtained in gamma camera images. The effect of In-111-BLMC in HNSCC xenografts was studied. In in vivo patient studies, we determined the tumor uptake of In-111-BLMC with gamma camera and the radioactivity from tumor samples using In-111-BLMC with specific activity of 75, 175, or 375 MBq/mg BLM. The S values, i.e. absorbed dose in a target organ per cumulated activity in a source organ, were simulated for In-111 and In-114m. In vitro studies showed the variation of sensitivity for external beam radiation, BLM, and In-111-BLMC between HNSCC cell lines. IC50 values for BLM were 1.6-, 1.8-, and 2.1-fold higher than In-111-BLMC (40 MBq/mg BLM) in three HNSCC cell lines. Specific In-111 activity of 40 MBq/mgBLM was more effective in killing cells than specific In-111 activity of 195MBq/mgBLM (p=0.0023). In-111-Cl3 alone had no killing effect. The percentage of cells in the G2/M phase increased after exposure to BLM and especially to In-111-BLMC in the three cell lines studied, indicating a G2/M block. The tumor-seeking behavior was shown in the in vivo imaging study of xenografted mice. BLM and In-111-BLMC were more effective than NaCl in reducing xenografted tumor size in HNSCC. The uptake ratios received from gamma images in the in vivo patient study varied from 1.2 to 2.8 in malignant tumors. However, the uptake of In-111-BLMC was unaffected by increasing the injected activity. A positive correlation existed between In-111-BLMC uptake, Ki-67/MIB activity, and number of mitoses. Regarding the S values, In-114m delivered a 4-fold absorbed radiation dose into the tumor compared with In-111, and thus, In-114m-BLMC might be more effective than In-111-BLMC at the DNA level. Auger-electron emitters, such as In-111 and In-114m, might have potential in the treatment of HNSCC. Further studies are needed to develop a radiopharmaceutical agent with appropriate physical properties of the radionuclide and a suitable carrier to bring it to the targeted tissue.