Quantitative Analysis of Whole-Mount Fluorescence-Stained Tumor Spheroids in Phenotypic Drug Screens.

Three-dimensional cell cultures, such as spheroids or organoids, serve as important models for drug screening purposes. Optical tissue clearing (OTC) enhances the visualization of fluorescence stainings and enables in toto microscopy of 3D cell culture models. Furthermore, subsequent automated image analysis tools convert qualitative confocal image sets into quantitative data. In this chapter, we describe a detailed protocol for preparation of HT29 cancer spheroids, 3D in toto immunostaining, glycerol-based OTC, whole-mount imaging, and semi-automated downstream image processing and segmentation for nuclear image analysis using open-source software.

Innovative Imaging Techniques: A Conceptual Exploration of Multi-Modal Raman Light Sheet Microscopy.

Advances in imaging of microscopic structures are supported and complemented by adaptive visualization tools. These tools enable researchers to precisely capture and analyze complex three-dimensional structures of different kinds such as crystals, microchannels and electronic or biological material. In this contribution, we focus on 3D cell cultures. The new possibilities can play a particularly important role in biomedical research, especially here in the study of 3D cell cultures such as spheroids in the field of histology. By applying advanced imaging techniques, detailed information about the spatial arrangement and interactions between cells can be obtained. These insights help to gain a better understanding of cellular organization and function and have potential implications for the development of new therapies and drugs. In this context, this study presents a multi-modal light sheet microscope designed for the detection of elastic and inelastic light scattering, particularly Rayleigh scattering as well as the Stokes Raman effect and fluorescence for imaging purposes. By combining multiple modalities and stitching their individual results, three-dimensional objects are created combining complementary information for greater insight into spatial and molecular information. The individual components of the microscope are specifically selected to this end. Both Rayleigh and Stokes Raman scattering are inherent molecule properties and accordingly facilitate marker-free imaging. Consequently, altering influences on the sample by external factors are minimized. Furthermore, this article will give an outlook on possible future applications of the prototype microscope.

Synthesis of large scale 3D microscopic images of 3D cell cultures for training and benchmarking.

The analysis of 3D microscopic cell culture images plays a vital role in the development of new therapeutics. While 3D cell cultures offer a greater similarity to the human organism than adherent cell cultures, they introduce new challenges for automatic evaluation, like increased heterogeneity. Deep learning algorithms are able to outperform conventional analysis methods in such conditions but require a large amount of training data. Due to data size and complexity, the manual annotation of 3D images to generate large datasets is a nearly impossible task. We therefore propose a pipeline that combines conventional simulation methods with deep-learning-based optimization to generate large 3D synthetic images of 3D cell cultures where the labels are known by design. The hybrid procedure helps to keep the generated image structures consistent with the underlying labels. A new approach and an additional measure are introduced to model and evaluate the reduced brightness and quality in deeper image regions. Our analyses show that the deep learning optimization step consistently improves the quality of the generated images. We could also demonstrate that a deep learning segmentation model trained with our synthetic data outperforms a classical segmentation method on real image data. The presented synthesis method allows selecting a segmentation model most suitable for the user's data, providing an ideal basis for further data analysis.

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward.

Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 µm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols - in particular, for screening purposes - clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

Increased cFLIP expression in thymic epithelial tumors blocks autophagy via NF-κB signalling.

The anti-apoptotic cellular FLICE-like inhibitory protein cFLIP plays a pivotal role in normal tissues homoeostasis and the development of many tumors, but its role in normal thymus (NT), thymomas and thymic carcinomas (TC) is largely unknown. Expression, regulation and function of cFLIP were analyzed in biopsies of NT, thymomas, thymic squamous cell carcinomas (TSCC), thymic epithelial cells (TECs) derived thereof and in the TC line 1889c by qRT-PCR, western blot, shRNA techniques, and functional assays addressing survival, senescence and autophagy. More than 90% of thymomas and TSCCs showed increased cFLIP expression compared to NT. cFLIP expression declined with age in NTs but not in thymomas. During short term culture cFLIP expression levels declined significantly slower in neoplastic than non-neoplastic primary TECs. Down-regulation of cFLIP by shRNA or NF-κB inhibition accelerated senescence and induced autophagy and cell death in neoplastic TECs. The results suggest a role of cFLIP in the involution of normal thymus and the development of thymomas and TSCC. Since increased expression of cFLIP is a known tumor escape mechanism, it may serve as tissue-based biomarker in future clinical trials, including immune checkpoint inhibitor trials in the commonly PD-L1high thymomas and TCs.

Transplanted fibroblasts proliferate in host bronchial tissue and enhance bronchial anastomotic healing in a rodent model.

INTRODUCTION: Healing of airway anastomoses after preoperative irradiation can be a significant clinical problem. The augmentation of bronchial anastomoses with a fibroblast-seeded human acellular dermis (hAD) was shown to be beneficial, although the underlying mechanism remained unclear. Therefore, in this study we investigated the fate of the fibroblasts transplanted to the scaffold covering the anastomosis. MATERIAL AND METHODS: 32 Fisher rats underwent surgical anastomosis of the left main bronchus. In a 2 × 2 factorial design, they were randomized to receive preoperative irradiation of 20 Gy and augmentation of the anastomosis with a fibroblast-seeded transplant. Fibroblasts from subcutaneous fat of Fischer-344 rat were transduced retrovirally with tdTomato for cell tracking. After 7 and 14 days, animals were sacrificed and cell concentration of transplanted and nontransplanted fibroblasts in the hAD as well as in the bronchial tissue was measured using RT-PCR. RESULTS: Migration of transplanted fibroblasts from dermis to bronchus were demonstrated in both groups, irradiated and nonirradiated. In the irradiated groups, there was a cell count of 7 × 104 ± 1 × 104 tomato+-fibroblasts in the bronchial tissue at day 7, rising to 1 × 105 ± 1 × 104 on day 14 (p <0.0001). Tomato+-cell concentration in hAD increased from 6 × 103 ± 1 × 103 at day 7 to 6 × 104 ± 1 × 104 at day 14 (p <0.0001). In the nonirradiated groups, tomato+-cell concentration in bronchus was 4 × 103 ± 1 × 103 on day 7 and 4 × 103 ± 1 × 103 at day 14. In the hAD tomato+ cell concentration rising from 1 × 104 ± 1 × 103 at day 7 to 2 × 104 ± 3 × 103 cells at day 14 (p = 0.0028). CONCLUSIONS: Transplanted fibroblasts in the irradiated groups proliferate and migrate into the irradiated host bronchial tissue, but not in the nonirradiated groups.

cFLIP overexpression in T cells in thymoma-associated myasthenia gravis.

OBJECTIVE: The capacity of thymomas to generate mature CD4+ effector T cells from immature precursors inside the tumor and export them to the blood is associated with thymoma-associated myasthenia gravis (TAMG). Why TAMG(+) thymomas generate and export more mature CD4+ T cells than MG(-) thymomas is unknown. METHODS: Unfixed thymoma tissue, thymocytes derived thereof, peripheral blood mononuclear cells (PBMCs), T-cell subsets and B cells were analysed using qRT-PCR and western blotting. Survival of PBMCs was measured by MTT assay. FAS-mediated apoptosis in PBMCs was quantified by flow cytometry. NF-κB in PBMCs was inhibited by the NF-κB-Inhibitor, EF24 prior to FAS-Ligand (FASLG) treatment for apoptosis induction. RESULTS: Expression levels of the apoptosis inhibitor cellular FLICE-like inhibitory protein (c-FLIP) in blood T cells and intratumorous thymocytes were higher in TAMG(+) than in MG(-) thymomas and non-neoplastic thymic remnants. Thymocytes and PBMCs of TAMG patients showed nuclear NF-κB accumulation and apoptosis resistance to FASLG stimulation that was sensitive to NF-κB blockade. Thymoma removal reduced cFLIP expression in PBMCs. INTERPRETATION: We conclude that thymomas induce cFLIP overexpression in thymocytes and their progeny, blood T cells. We suggest that the stronger cFLIP overexpression in TAMG(+) compared to MG(-) thymomas allows for the more efficient generation of mature CD4+ T cells in TAMG(+) thymomas. cFLIP overexpression in thymocytes and exported CD4+ T cells of patients with TAMG might contribute to the pathogenesis of TAMG by impairing central and peripheral T-cell tolerance.

In vivo Quantification of the Effects of Radiation and Presence of Hair Follicle Pores on the Proliferation of Fibroblasts in an Acellular Human Dermis in a Dorsal Skinfold Chamber: Relevance for Tissue Reconstruction following Neoadjuvant Therapy.

INTRODUCTION: In neoadjuvant therapy, irradiation has a deleterious effect on neoangiogenesis. The aim of this study was to examine the post-implantation effects of neoadjuvant irradiation on the survival and proliferation of autologous cells seeded onto an acellular human dermis (hAD; Epiflex). Additionally, we examined the influence of dermal hair follicle pores on viability and proliferation. We used dorsal skinfold chambers implanted in rats and in-situ microscopy to quantify cell numbers over 9 days. METHODS: 24 rats received a skinfold chamber and were divided into 2 main groups; irradiated and unirradiated. In the irradiated groups 20Gy were applied epicutaneously at the dorsum. Epiflex pieces were cut to size 5x5mm such that each piece had either one or more visible hair follicle pores, or no such visible pores. Fibroblasts were transduced lentiviral with a fluorescent protein for cell tracking. Matrices were seeded statically with 2.5x104 fluorescent fibroblasts and implanted into the chambers. In each of the two main groups, half of the rats received Epiflex with hair follicle pores and half received Epiflex without pores. Scaffolds were examined in-situ at 0, 3, 6 and 9 days after transplantation. Visible cells on the surface were quantified using ImageJ. RESULTS: In all groups cell numbers were decreased on day 3. A treatment-dependent increase in cell numbers was observed at subsequent time points. Irradiation had an adverse effect on cell survival and proliferation. The number of cells detected in both irradiated and non-irradiated subjects was increased in those subjects that received transplants with hair follicle pores. DISCUSSION: This in-vivo study confirms that radiation negatively affects the survival and proliferation of fibroblasts seeded onto a human dermis transplant. The presence of hair follicle pores in the dermis transplants is shown to have a positive effect on cell survival and proliferation even in irradiated subjects.

Effect of dynamic seeding methods on the distribution of fibroblasts within human acellular dermis.

The purpose of this investigation was to compare different dynamic cell seeding methods regarding their seeding efficiency, homogeneity, infiltration depth and proliferation within a human acellular dermis. In addition, the growth behaviour was observed during a 12-day static in vitro culture. The dynamic methods included orbital-shaker seeding and the use of a plate centrifuge with different rotational speeds, combinations of low-pressure for matrix degassing and centrifugal seeding. Scaffolds were incubated for up to 12 days statically. Cell distribution and infiltration depth were analysed histologically at days 0, 4, 8 and 12. Seeding efficiency and cell proliferation were quantified with the MTT-assay at the same time points. Centrifugal seeding with 300g for 5 × 1 min combined with matrix degassing significantly increased the seeding efficiency and homogeneity compared to the other methods. However, following static culture, no cells were detectable after 4 days in the inner matrix zones. Furthermore, none of the degassing+centrifugation groups reached a significantly higher proliferation at day 8 compared to the reference. The use of a single dynamic method resulted in an inefficient cell seeding. We archived the highest seeding efficiency, homogeneity and infiltration depth using a combination of degassing+centrifugation at 300g for 5 × 1 min.

Effect on the tensile strength of human acellular dermis (Epiflex®) of in-vitro incubation simulating an open abdomen setting.

BACKGROUND: The use of human acellular dermis (hAD) to close open abdomen in the treatment process of severe peritonitis might be an alternative to standard care. This paper describes an investigation of the effects of fluids simulating an open abdomen environment on the biomechanical properties of Epiflex® a cell-free human dermis transplant. METHODS: hAD was incubated in Ringers solution, blood, urine, upper gastrointestinal (upper GI) secretion and a peritonitis-like bacterial solution in-vitro for 3 weeks. At day 0, 7, 14 and 21 breaking strength was measured, tensile strength was calculated and standard fluorescence microscopy was performed. RESULTS: hAD incubated in all five of the five fluids showed a decrease in mean breaking strength at day 21 when compared to day 0. However, upper GI secretion was the only incubation fluid that significantly reduced the mechanical strength of Epiflex after 21 days of incubation when compared to incubation in Ringer's solution. CONCLUSION: hAD may be a suitable material for closure of the open abdomen in the absence of upper GI leakage and pancreatic fistulae.

Human acellular dermis seeded with autologous fibroblasts enhances bronchial anastomotic healing in an irradiated rodent sleeve resection model.

BACKGROUND: The combination of neoadjuvant radiochemotherapy and parenchyma-preserving sleeve resection for lung cancer remains controversial because of potentially increased rate of anastomotic breakdown. We analyzed the effects of applying a decellularized human dermis transplant seeded with autologous fibroblasts in a rodent sleeve resection model with neoadjuvant radiotherapy. MATERIALS AND METHODS: A total of 64 male Fisher rats underwent a transsection and surgical anastomosis of the left main bronchus and were randomized to receive plus/minus radiation treatment and plus/minus augmentation of the anastomosis with a fibroblast-seeded dermis transplant (2 × 2 factorial design). A µCT scan was performed at postoperative days 7 and 14, and the animals were sacrificed on day 14. Anastomotic bursting pressure and hydroxyproline concentration were measured. RESULTS: In the irradiated groups, the anastomotic bursting pressure was significantly higher in the augmented group at day 7 (100.9 ± 18.3 vs 141.3 ± 18.0 kPa, p = 0.0005) but not at day 14. Hydroxyproline levels showed a similar pattern in the irradiated group with significant differences at day 7 (7 days postoperative 158 ± 11.6 vs 198.2 ± 10.9 nmol/mg, p < 0.0001) but not at day 14 postoperatively. CONCLUSIONS: Augmentation of a bronchial anastomosis by a dermal matrix, seeded with autologous, viable fibroblasts improves early wound breaking strength. Fibroblast-enhanced dermal matrices provide a new and easily usable tool to prevent early anastomotic leakage after neoadjuvant chemoradiation in locally advanced lung cancer.

Effect of static seeding methods on the distribution of fibroblasts within human acellular dermis.

INTRODUCTION: When developing tissue engineered solutions for existing clinical problems, cell seeding strategies should be optimized for desired cell distribution within matrices. The purpose of this investigation was to compare the effects of different static cell seeding methods and subsequent static cell culture for up to 12 days with regard to seeding efficiency and resulting cellular distribution in acellular dermis. MATERIALS AND METHODS: The seeding methods tested were surface seeding of both unmodified and mechanically incised dermis, syringe injection of cell suspension, application of low-pressure and use of an ultrasonic bath to remove trapped air. The effect of "platelet derived growth factor" (PDGF) on surface seeding and low pressure seeding was also investigated. Scaffolds were incubated for up to 12 days and were histologically examined at days 0, 4, 8 and 12 for cell distribution and infiltration depth. The metabolic activity of the cells was quantified with the MTT assay at the same time points. RESULTS: The 50 ml syringe degassing procedure produced the best results in terms of seeding efficiency, cell distribution, penetration depth and metabolic activity within the measured time frame. The injection and ultrasonic bath methods produced the lowest seeding efficiency. The incision method and the 20 ml syringe degassing procedure produced results that were not significantly different to those obtained with a standard static seeding method. CONCLUSION: We postulate that air in the pores of the human acellular dermis (hAD) hinders cell seeding and subsequent infiltration. We achieved the highest seeding efficiency, homogeneity, infiltration depth and cell growth within the 12 day static culturing period by degassing the dermis using low- pressure created by a 50 ml syringe. We conclude that this method to eliminate trapped air provides the most effective method to seed cells and to allow cell proliferation in a natural scaffold.

Confocal laser scanning microscopy evaluation of an acellular dermis tissue transplant (Epiflex®).

The structure of a biological scaffold is a major determinant of its biological characteristics and its interaction with cells. An acellular dermis tissue transplant must undergo a series of processing steps, to remove cells and genetic material and provide the sterility required for surgical use. During manufacturing and sterilization the structure and composition of tissue transplants may change. The composition of the human cell-free dermis transplant Epiflex® was investigated with specific attention paid to its structure, matrix composition, cellular content and biomechanics. We demonstrated that after processing, the structure of Epiflex remains almost unchanged with an intact collagen network and extracellular matrix (ECM) protein composition providing natural cell interactions. Although the ready to use transplant does contain some cellular and DNA debris, the processing procedure results in a total destruction of cells and active DNA which is a requirement for an immunologically inert and biologically safe substrate. Its biomechanical parameters do not change significantly during the processing.

Differential survival of AML subpopulations in NOD/SCID mice.

OBJECTIVE: Leukemia-initiating cells can retrospectively be defined by tumorigenicity in immunodeficient mice and be characterized by surface markers. The latter still being discussed for acute myeloid leukemia (AML), nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were used to evaluate long-time reconstitution and expansion of AML subpopulations. MATERIALS AND METHODS: Bone marrow cells from patients with AML were separated according to CD34 expression, aldehyde dehydrogenase (ALDH) activity, and divisional kinetics in comparison to cord blood-derived CD34(+) hematopoietic stem cells, evaluating survival and expansion in NOD/SCID mice. The AML long-term surviving capacity of subpopulations recovered from NOD/SCID mice was confirmed by ex vivo survival. RESULTS: AML mononuclear cells were detected in bone marrow and spleen of NOD/SCID mice 12 weeks after transplantation. The majority of recovered cells were CD34(+) and significantly more CD34(+) cells were recovered after application of ALDH(bright) (high ALDH activity), CD34(+), or slowly dividing (PKH(bright)) than after ALDH(dim), CD34(-), or fast dividing (PKH(dim)) cell application. CD123(+), CD63(+), and CD44v7(+) cells were also more abundant after the transfer of ALDH(bright) or CD34(+) AML mononuclear cells. In the spleen, large AML cell clusters were only recovered after ALDH(bright), CD34(+), or PKH(bright) cell transfer. Importantly, in secondary long-term in vitro cultures, quite exclusively CD34(+) AML mononuclear cells survived and expanded. CONCLUSIONS: Separation of ALDH(bright), CD34(+), or PKH(bright) cells enriches for AML long-term surviving capacity, which reside in the CD34(+) subpopulation, as rather exclusively CD34(+) cells survived and expanded in vivo and ex vivo. Long-term survival capacity may be supported by CD44v7 expression.

Thymocyte expansion and maturation: crosstalk of CD44v6 on thymocytes and panCD44 on stroma cells.

Re-acquisition of immunocompetence after allogeneic bone marrow cell (BMC) transplantation depends on intrathymic maturation of the allogeneic T progenitor cells. We recently reported that CD44 promotes progenitor homing into the thymus and T-cell maturation and now elucidate the molecular mechanisms of CD44-supported thymocyte maturation. Lethally irradiated, tumor-bearing mice, allogeneically reconstituted with T-cell-depleted BMC and a small number of common lymphoid progenitor 2 cells (CLP2) from transgenic (TG) mice, that express ratCD44v4-v7 under the Thy1 promoter, showed accelerated immunocompetent T-cell recovery compared with mice reconstituted with non-transgenic (NTG) CLP2. In addition, graft-versus-host disease was strongly reduced after tumor vaccination. TG, but not NTG double-negative (DN) thymocytes showed high proliferative potential, accompanied by constitutive association of lck with CD44. Importantly, when thymocyte adhesion was strengthened by anti-CD44, co-cultures of DN thymocytes with thymic stroma supported DN thymocyte maturation. The close contact between DN thymocytes and thymic stroma promoted persisting activation of lck and ERK1/2, particularly in CD44v6(+) DN thymocytes. Thus, intrathymic T-cell maturation in allogeneically reconstituted, leukemia-bearing hosts can be considerably accelerated by high CD44v6 expression in early thymocytes, in which proliferation-supporting signals are initiated by a crosstalk between CD44v6 on thymocytes and panCD44 on the thymic stroma.

Improved T and B cell recovery by the transfer of slowly dividing human hematopoietic stem cells.

Human hematopoietic stem cells giving rise to long term initiating cells in vitro are enriched in a CD34(+) slow dividing fraction (SDF). Here, we tested reconstitution and multilineage differentiation of this CD34(+) SDF in NOD/SCID mice. In the bone marrow a slightly higher percentage of human hematopoietic progenitors were recovered after the transfer of the SDF compared to the fast dividing fraction. Instead, T cell maturation in the rudimentary thymus and lymph node repopulation was only initiated by the SDF. The capacity of the SDF to differentiate and mature in the patients' thymus could provide an advantage in immunocompetence recovery.

CD44v6 dependence of premetastatic niche preparation by exosomes.

The metastasizing capacity of the rat pancreatic adenocarcinoma BSp73ASML (ASML(wt)) is strikingly reduced by a knockdown of CD44v4-v7 (ASML(kd)). We used this model to analyze the role of the CD44 variant isoform (CD44v) in (pre)metastatic niche formation. Intrafootpad injections of ASML(wt)-, but not ASML(kd)-conditioned medium (CM), strongly promote settlement of ASML(kd) cells in lymph nodes and lung. Fractionation of CM revealed a contribution by a soluble matrix and exosomes, where the CD44v6-containing ASML(wt)-soluble fraction can complement ASML(kd)-exosomes, but not vice versa. This implies that exosomes are the final actors, are CD44v-independent, but require a soluble matrix, which depends on CD44v. Analyzing the composition revealed that only the ASML(wt)-matrix contains c-Met and urokinase-type plasminogen activator receptor. In vitro, mostly ASML(wt)-exosomes promote proliferation and induce gene expression in metastatic organ cells. However, in vivo corresponding changes in the (pre) metastatic organ are only observed when both, exosomes plus the soluble matrix, are provided. Thus, neither CD44v nor exosomes alone suffice for (pre)metastatic niche formation. Instead, CD44v suffices for assembling a soluble matrix, which allows exosomes, independent of their origin from poorly or highly metastatic cells, to modulate (pre) metastatic organ cells for tumor cell embedding and growth.

The efficacy of an IL-1alpha vaccine depends on IL-1RI availability and concomitant myeloid-derived suppressor cell reduction.

We recently reported that tumor-derived interleukin (IL)-1beta strongly promotes tumor growth by inducing myeloid-derived suppressor cell (MDSC) and regulatory T-cell (T(reg)) expansion. To see whether redirection of an immune response can be achieved through immune response-supporting IL-1alpha application, IL-1RI competent (IL-1RI(comp)) and IL-1RI-deficient (IL-1RI(-/-)) mice received IL-1alpha cDNA-transformed attenuated Salmonella typhimurium (SL-IL-1alpha) and/or lysates from methycholanthrene-induced IL-1(comp) or IL-1(-/-) fibrosarcoma cells. Vaccination with SL-IL-1alpha and/or tumor lysate exerted only a minor effect on the survival of IL-1alpha/beta(-/-) and none on IL-1alpha(comp) tumor-bearing mice despite induction of a potent antitumor response, that was overridden by intratumoral and systemic expansion of MDSC. Application of all-trans-retinoic acid together with anti-CD25 efficiently coped with MDSC and T(reg) expansion. Vaccination concomitantly with application of all-trans-retinoic acid and anti-CD25 treatment significantly increased the survival time and rate of IL-1alpha/beta(comp), but even of IL-1alpha(-/-)beta(comp) IL-1RI(comp) tumor-bearing mice. Instead, in IL-1RI(-/-) mice, though MDSC expansion was weaker, SL-IL-1alpha application hardly displayed any therapeutic efficacy, which implies signal transduction through IL-1alpha binding to the IL-1RI as an essential component for immune response induction. Taken together, IL-1alpha can efficiently support tumor vaccination, as far as expansion of MDSC and T(reg) is controlled. However, care should be taken to interfere with MDSC expansion/activation not through a blockade of the IL-1RI, which is the preferential target of IL-1alpha.

CD44 promotes progenitor homing into the thymus and T cell maturation.

Regain of immunocompetence after myeloablation and bone marrow cell (BMC) reconstitution essentially depends on T progenitor homing into the thymus and intrathymic T cell maturation. CD44 facilitates progenitor homing and settlement in the bone marrow and is known as a T progenitor marker. In search for improving regain of immunocompetence after BMC reconstitution, we explored whether the CD44 standard (CD44 s) and/or variant isoforms CD44v6 and CD44v7 contribute to thymus repopulation and thymocyte maturation. Antibody-blocking studies and cells/mice with a targeted deletion of CD44v6/7 or CD44v7 revealed that CD44s, but not CD44v6 and CD44v7, has a major impact on progenitor cell homing into the thymus. Instead, CD44v6 strengthens apoptosis resistance and expansion of early thymocytes. CD44v6-induced apoptosis resistance, most strong in double-negative (DN) thymocytes, is accompanied by Akt activation. CD44v6-induced proliferation of DN cells proceeds via activation of the MAPK pathway. At later stages of T cell maturation, CD44 acts as an accessory molecule, initiating and supporting TCR/CD3 complex-mediated signal transduction in double-positive and single-positive thymocytes. Thus, CD44 plays a major role in thymus homing. In addition, CD44v6 is important for survival and expansion of early thymocytes. These findings suggest that strengthening CD44v6 expression on lymphoid progenitors could well contribute to accelerated regain of immunocompetence.

Tumorigenicity of IL-1alpha- and IL-1beta-deficient fibrosarcoma cells.

Analyzing the growth of fibrosarcoma lines derived from IL-1alpha-, IL-1beta- , or IL-1alphabeta-knockout (-/-) mice in the immunocompetent host revealed that tumor-derived IL-1alpha and IL-1beta exert strong and opposing effects on immune response induction, which prohibited the evaluation of a potential impact on tumorigenicity. Therefore, in vivo growth of IL-1-deficient tumor lines was evaluated in nu/nu mice and was compared with in vitro growth characteristics. All IL-1-deficient fibrosarcoma lines grow in immunocompromised mice. However, IL-1alpha(-/-)beta-competent (comp) lines grow more aggressively, efficiently induce angiogenesis, and recruit inflammatory cells. Despite stronger tumorigenicity of IL-1beta(comp) lines, IL-1alpha strengthens anchorage-independent growth, but both IL-1alpha and IL-1beta support drug resistance. Corresponding to the aggressive growth, IL-1beta(comp) cells display increased matrix adhesion, motility, and cable formation on matrigel, likely supported by elevated alpha(v)/beta3 and matrix metalloroteinase expression. Recruitment of myeloid cells requires IL-1beta but is regulated by IL-1alpha, because inflammatory chemokine and cytokine expression is stronger in IL-1alpha(-/-)beta(comp) than in IL-1(wt) lines. This regulatory effect of tumor-derived IL-1alpha is restricted to the tumor environment and does not affect systemic inflammatory response induction by tumor-derived IL-1beta. Both sarcoma cell-derived IL-1alpha and IL-1beta promote tumor growth. However, IL-1alpha exerts regulatory activity on the tumor cell-matrix cross-talk, and only IL-1beta initiates systemic inflammation.