Adherent Cell Population Research Articles

Abstract 3783: Effective MEK inhibitor combinations to target tumor heterogeneity in EGFR mutant transformed SCLC

Abstract Background: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective for the treatment of EGFR-mutant non-small lung cancer (NSCLC). In almost all cases, however, the response duration is limited and resistance ultimately emerges. Transformation of NSCLC to small cell lung cancer (SCLC) occurs as one of the resistance mechanisms to EGFR-TKIs. To date, the mechanism associated with this transformation is largely unknown, necessitating the need to establish an effective treatment strategy for patients with transformed SCLC (tSCLC). Methods: We isolated cancer cells that grow as floating aggregates (F) and adherent monolayers (Ad) from the pleural effusion of three patients with tSCLC to establish patient derived cell lines (DFCI112F, DFI112ad, DFCI190F, DFCI190ad, DFCI283F, DFCI283ad). In parallel, patient derived xenograft (PDX) models from these patients were successfully established by subcutaneous implantation of pleural effusions in NSG mice. Transcriptomic and genomic characterization of the cell lines were performed using whole-exome sequencing (WES) and RNA sequencing. These cell lines were further treated with a panel of agents, to screen for effective combination therapies. Results: Biological characterization of the created cell lines revealed that the floating aggregates have neuroendocrine (NE) features, while the adherent cell population presents non-NE/mesenchymal characteristics. The histology of the PDX tumors was confirmed as SCLC. According to WES, all tSCLC cell lines retained the original activating EGFR mutations. RB1 and TP53 were universally inactivated. Co-culturing the floating-NE cells with the adherent non-NE cells promoted the proliferation of the floating-NE cells in vitro. NE cells (DFCI112F) and non-NE cells (DFCI112ad) were injected into nude mice either as single or admixed populations. Tumor growth was observed only in admixed cell populations. In an effort to target both NE and non-NE populations, we performed a drug screen and demonstrated that MEK inhibitors are selectively efficacious against non-NE populations. We further evaluated the MEK inhibitor combination therapies with agents targeting NE populations (cisplatin, ABT-263 and JQ1), which effectively eliminated both NE and non-NE components in vitro. Our further study demonstrated that ABT-263 in combination with MEK inhibitors presented a synergistic effect, which could be a promising therapeutic combination for tSCLC patients. Conclusion: We demonstrate that the cancer cell lines established from tSCLC patients consist of heterogeneous population of cells with NE and non-NE/mesenchymal characteristics. The crosstalk between these two cell populations is observed and our findings underscore the importance of targeting tumor heterogeneity as a promising treatment strategy for patients with tSCLC. Citation Format: Atsuko Ogino, Xinmeng Jasmine Mu, Mika Lin, Antonio Calles, Stephen Wang, Man Xu, Lynette M. Scholl, Geoffrey R. Oxnard, Paul Kirschmeier, Pasi A. Jänne. Effective MEK inhibitor combinations to target tumor heterogeneity in EGFR mutant transformed SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3783.

Adherent muscle connective tissue fibroblasts are phenotypically and biochemically equivalent to stromal fibro/adipogenic progenitors

Extracellular matrix (ECM) gives structure, support, and is the niche for several cells found in skeletal muscle. ECM is mainly produced by muscle connective tissue (CT) fibroblasts during development and regeneration. Stromal fibroadipogenic progenitors (FAPs) are CT fibroblasts-like mesenchymal progenitors (MPs) with important roles in regeneration and degeneration. Chronic damage restrains the normal regenerative behavior of muscle fibroblasts/FAPs. Thus, the isolation and study of these mesenchymal progenitors are of crucial importance for understanding their behavior and biology. We investigated whether adult muscle CT fibroblasts (hereafter referred to as adherent fibroblasts [aFbs]) cultured via pre-plating strategy belong to a heterogeneous population of FAPs. By combining microscopy, western blot analyses, flow cytometry, and FACS we determined that aFbs isolated from skeletal muscle largely overlap with FAPs. In addition, we used the PDGFRαEGFP mice in order to corroborate our results with EGFP+ FAPs. Moreover, our strategy allows the isolation of activated EGFP+ FAPs from the murine DMD model PDGFRαEGFP; mdx and PDGFRαEGFP denervated mice. Here we report that 1 h 30 min of pre-plating strategy allows the isolation and culture of a highly enriched population of aFbs. These cells are phenotypically and biochemically a FAPs-like population of adherent cells. In addition, aFbs respond in the same fashion as FAPs to Nilotinib, an inducer of FAPs apoptosis. Moreover, flow cytometry characterization of these aFbs suggests that 85% of them express the MP marker PDGFRα, and isolation of aFbs from the PDGFRαEGFP mice suggests that 75% of them show high EGFP expression. Furthermore, TGF-β1 induces aFbs proliferation, myofibroblast differentiation, and ECM production. We were also able to isolate activated aFbs from skeletal muscle of the DMD mice and from the PDGFRαEGFP mice 2-days after denervation. Our findings suggest that the in vitro pre-plating strategy allows the isolation and culture of a relatively pure aFbs population, which resembles FAPs in vitro.

Competitive interaction on dual-species biofilm formation by spoilage bacteria, Shewanella baltica and Pseudomonas fluorescens.

This study aims to characterize the biofilm produced by mono- and dual-species of Shewanella baltica and Pseudomonas fluorescens as fish spoilers at the different incubation temperature, and to elucidate the interactive behaviour of dual-species biofilm development. The mono- and dual-species biofilm formation and adhesion characteristics of S. baltica and P. fluorescens were evaluated by using crystal violet staining, scanning electron microscopy and confocal laser scanning microscopy. Results showed that P. fluorescens had significantly higher biofilm biomass and polysaccharides production than S. baltica, and two isolates reached the maximum biofilm biomass during the early stationary phase. Lower biomass and polysaccharides in dual-species biofilms were observed compared to mono-species of P. fluorescens. Meanwhile, S. baltica and P. fluorescens formed fragile and viscous pellicles with different spatial architectures respectively. In dual-species pellicle few large microcolonies were dominated by P. fluorescens. Compared to mono-species of PF07, adherent cell population and biofilm thickness at the developing phase significantly decreased, and biofilm-forming cycle prolonged in the dual-species biofilms. Biofilm formation and adhesion of mono- and dual-species at 4 or 15°C were significantly higher than at 30°C during the same phase. The culture supernatant extracts of the two spoilage strains greatly inhibited biofilm development to each other. Shewanella baltica and P. fluorescens had different biofilm and pellicle characteristics, and the inhibitory development on dual-species biofilm was associated with the competitive interaction by the two psychrotrophic spoilage bacteria. This work contributes to a better understanding of interactive behaviour of multispecies biofilm communities by psychrotrophic spoilage bacteria at low temperature, which could contribute to further control contamination of spoilage organism during the preservation and processing of aquatic products.

Adhesion of Thymocytes to the Thymic Epithelial Cells and Participation of Neyropilin-1 and Plexin A1 in the Adhesion

Thymic epithelial cells play not only an important structural role, but also create the microenvironment necessary for the maturation and differentiation of thymocytes. One of the stages of maturation of thymocytes is their adhesion to the epithelial cells of the thymus, the mechanism of which has not been sufficiently studied. The goal of the study was investigation of the interaction of mouse thymocytes with cortical cTEC1-2 and the medullary mTEC3-10 thymic epithelial cell lines, namely, the dynamics of the adhesion, apoptosis, and population composition, as well as the expression of the neuropilin-1 (Nrp-1) and plexin A1 receptors (PlexA1) among adherent, nonadherent, and control cells cultured without epithelium. The maximal adhesion index was observed for a 30-min cocultivation; after this time, the index decreased. The main population of adherent cells consisted from immature CD4+CD8+ lymphocytes, and the relative content of mature CD4+ and CD8+ thymocytes was lower than in the control. We have shown for the first time that, even at short cocultivation periods (30 and 120 min), an increase in the content of thymocytes at the early apoptosis phase was observed among the population of adherent cells. For the first time, data have been obtained demonstrating the participation of Nrp-1 and PlexA1 in the process of thymocyte adhesion to mouse thymic epithelial cells as independent molecules in the absence of their semaphorin 3A ligand. It was shown that the expression of Nrp-1 on the surface of adherent thymocytes was significantly reduced during the adhesion process compared to the control, and the expression of PlexA1 increased. Preincubation of thymocytes with antibodies to Nrp-1 increased their adhesion to epithelial cells. The data obtained contribute to a better understanding of intercellular relationships in the thymus gland.

The majority of cells in so-called “mesenchymal stem cell” population are neither stem cells nor progenitors

The first-passage adherent human bone marrow fibroblast-like cell population corresponds, in terms of phenotype and three-lineage differentiation capacity (assayed in bulk culture), to commonly termed "mesenchymal stem cells". Here we determine the proportion of high proliferative capacity multipotent cells present in this population in order to estimate the proportion of cells that can or cannot be considered as stem and progenitor cells. The single-cell cultures were established starting from human bone marrow-derived first-passage fibroblast-like cells and the proliferating clones were either transferred to secondary cultures to evaluate their further clonogenicity, or split into three wells to assess differentiation into each of the three different lineages. The analysis of 197 single-cell cultures from three different bone marrow donors shows that only∼40% of so-called "mesenchymal stem cells" exhibit multipotency and are capable of sustained clonogenicity in secondary cultures. Even in the first ex vivo passage under favorable conditions the majority (∼60%) of so-called "mesenchymal stem cells" are not multipotent and thus do not represent a stem cell entity.

Daily oscillations in expression and responsiveness of Toll-like receptors in splenic immune cells

Circadian rhythms refer to biologic processes that oscillate with an approximate 24-h period. These rhythms direct nearly all aspects of animal behavior and physiology. The aim of our study was to determine if Toll-like receptor (TLR) expression and responsiveness exhibit time-of-day dependent differences. Therefore, we isolated an adherent splenocyte population, which consisted primarily of B cells, dendritic cells, and macrophages, over the course of a 24-h light-dark period and measured daily changes in Tlr1-8 mRNA levels and cytokine expression after cells were challenged at Zeitgeber time (ZT) 1 or ZT13 with a TLR ligand. In addition, we assessed TLR3 protein levels in adherent splenocytes over the 24-h light-dark period and challenged mice at ZT1 or ZT13 with poly(I:C), the TLR3 ligand. Our study revealed that in this adherent cell population, all Tlrs exhibited rhythmic expression except Tlr2 and Tlr5, and all TLRs, except TLR8, demonstrated daily variations in responsiveness after challenge with their respective ligand. We also revealed that TLR3 protein levels fluctuate over the daily light-dark cycle in adherent splenocytes and mice exhibit a time-of-day dependent immune response when challenged with poly(I:C). Finally, we demonstrated that mRNA levels of Tlr2 and Tlr6 display rhythmic expression in splenic macrophages. Taken together, these findings could have important implications for TLR-directed therapeutics.

Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method.

12–14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs' purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P < 0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

Rapid enrichment of adipose derived mesenchymal stromal cells for use in cellular therapies

Background: Mesenchymal stromal cells (MSC) are multipotent cells that can be isolated from many tissues and are the subject of multiple clinical investigations. While adipose tissue is a reliable source of MSC, it still requires enrichment to increase potency and decrease volume prior to use in autologous cellular therapy. Prolonged in vitro culture can enrich and expand MSC but may also induce functional changes and stem cell senescence. Methods: The stromal vascular fraction (SVF) obtained by enzymatic digestion of lipoaspirated human adipose tissue was plated at a maximum density of 100,000–200,000 cells/cm2 onto tissue culture flasks. A method which involved washing every 24 hours to remove non-adherent cells (Method A) was compared to a method that involved harvesting and re-plating cells in fresh cell culture flasks (Method B). Adherent cell populations containing the purified MSC could then be harvested and assayed at various time points. Results: MSC from the SVF were enriched from 16% of cells positive for CD73 at day 0 to 39% at day 1 to 59% within 2 days, with minimal (1–2 cell doublings) cellular expansion for Method B. Flow cytometric analysis demonstrated co-expression of known MSC phenotypic markers (CD73, CD105, and CD90) along with maintenance of functional capabilities (e.g., cell growth, colony formation and directed differentiation). After enrichment MSC can be harvested and concentrated for resuspension in minimal volumes for clinical use. Discussion: The maximum density plating and short-term culture approach described herein represents a simple and novel method to rapidly isolate MSC for clinical therapies, with minimal costs and time, which can also be performed in a closed culture system.

Highly efficient cellular cloning using Ferro-core Micropallet Arrays

Advancing knowledge of biological mechanisms has come to depend upon genetic manipulation of cells and organisms, relying upon cellular cloning methods that remain unchanged for decades, are labor and time intensive, often taking many months to come to fruition. Thus, there is a pressing need for more efficient processes. We have adapted a newly developed micropallet array platform, termed the “ferro-core micropallet array”, to dramatically improve and accelerate the process of isolating clonal populations of adherent cells from heterogeneous mixtures retaining the flexibility of employing a wide range of cytometric parameters for identifying colonies and cells of interest. Using transfected (retroviral oncogene or fluorescent reporter construct) rat 208 F cells, we demonstrated the capacity to isolate and expand pure populations of genetically manipulated cells via laser release and magnetic recovery of single micropallets carrying adherent microcolonies derived from single cells. This platform can be broadly applied to biological research, across the spectrum of molecular biology to cellular biology, involving fields such as cancer, developmental, and stem cell biology. The ferro-core micropallet array platform provides significant advantages over alternative sorting and cloning methods by eliminating the necessity for repetitive purification steps and increasing throughput by dramatically shortening the time to obtain clonally expanded cell colonies.

In Vitro Response of Human Peripheral Blood Mononuclear Cells (PBMC) to Collagen Films Treated with Cold Plasma.

The implantation of biomedical devices, including collagen-based implants, evokes an inflammatory response. Despite inflammation playing an important role in the early stages of wound healing, excessive and non-resolving inflammation may lead to the poor performance of biomaterial implants in some patients. Therefore, steps should be taken to control the level and duration of an inflammatory response. In this study, oxygen and nitrogen gas plasmas were employed to modify the surface of collagen film, with a view to modifying the surface properties of a substrate in order to induce changes to the inflammatory response, whilst maintaining the mechanical integrity of the underlying collagen film. The effects of cold plasma treatment and resultant changes to surface properties on the non-specific inflammatory response of the immune system was investigated in vitro in direct contact cell culture by the measurement of protein expression and cytokine production after one and four days of human peripheral blood mononuclear cell (PBMC) culture. The results indicated that compared to oxygen plasma, nitrogen plasma treatment produced an anti-inflammatory effect on the collagen film by reducing the initial activation of monocytes and macrophages, which led to a lower production of pro-inflammatory cytokines IL-1β and TNFα, and higher production of anti-inflammatory cytokine IL-10. This was attributed to the combination of the amino chemical group and the significant reduction in roughness associated with the introduction of the nitrogen plasma treatment, which had an effect on the levels of activation of the adherent cell population.

Patterning of sharp cellular interfaces with a reconfigurable elastic substrate.

Micropatterned cocultures are a useful experimental tool for the study of cell-cell interactions. Patterning methods often rely on sequential seeding of different cell types or removal of a barrier separating two populations, but it is difficult to pattern sharp interfaces between pure populations with low cross-contamination when using these approaches. Patterning by the use of reconfigurable substrates can overcome these limitations, but such methods can be costly and challenging to employ in a typical biology laboratory. Here, we describe a low-cost and simple-to-use reconfigurable substrate comprised of a transparent elastic material that is partially cut to form a slit that opens when the device is stretched. The slit seals back up when released, allowing two initially separate, adherent cell populations to be brought together to form a contact interface. Fluorescent imaging of patterned cocultures demonstrates the early establishment of a sharp cellular interface. As a proof of principle, we demonstrate the use of this device to study competition at the interface of two stem cell populations.

Lab-to-clinic application of stem cell therapy for stroke

Stem cell therapy or “cell therapy” has been demonstrated to be a potent treatment intervention in animal models of acute ischemic stroke, and recently has been introduced as an experimental therapy in early phase clinical trials. Among the many stem cells, the bone marrow adherent cell type known as mesenchymal stem cells have emerged in laboratory studies as a safe and effective therapy for ischemic stroke and other brain diseases. In particular, a unique population of adherent bone marrow-derived cells, called MultiStem cells, display immunomodulatory effects and are a promising allogeneic cell therapy in acute ischemic stroke. Here, we describe the preclinical evidence supporting the use of MultiStem in the acute setting of ischemic stroke and the translation into an early phase clinical trial in ischemic stroke.

A convenient and effective strategy for the enrichment of tumor-initiating cell properties in prostate cancer cells.

Stem-like prostate cancer (PrCa) cells, also called PrCa stem cells (PrCSCs) or PrCa tumor-initiating cells (PrTICs), are considered to be involved in the mediation of tumor metastasis and may be responsible for the poor prognosis of PrCa patients. Currently, the methods for PrTIC sorting are mainly based on cell surface marker or side population (SP). However, the rarity of these sorted cells limits the investigation of the molecular mechanisms and therapeutic strategies targeting PrTICs. For PrTIC enrichment, we induced cancer stem cell (CSC) properties in PrCa cells by transducing three defined factors (OCT3/4, SOX2, and KLF4), followed by culture with conventional serum-containing medium. The CSC properties in the transduced cells were evaluated by proliferation, cell cycle, SP assay, drug sensitivity technology, in vivo tumorigenicity, and molecular marker analysis of PrCSCs compared with parental cells and spheroids. After culture with serum-containing medium for 8days, the PrCa cells transduced with the three factors showed significantly enhanced CSC properties in terms of marker gene expression, sphere formation, chemoresistance to docetaxel, and tumorigenicity. The percentage of CD133+/CD44+ cells was ninefold higher in the transduced cell population than in the adherent PC3 cell population (2.25 ± 0.62 vs. 0.25 ± 0.12%, respectively), and the SP increased to 1.22 ± 0.18% in the transduced cell population, but was undetectable in the adherent population. This method can be used to obtain abundant PrTIC material and enables a complete understanding of PrTIC biology and development of novel therapeutic agents targeting PrTICs.

Mesenchymal stromal cells: potential roles in graft‐versus‐host disease prophylaxis and treatment

Mesenchymal stromal cells: potential roles in graft‐versus‐host disease prophylaxis and treatment

In Situ Analysis of Small Populations of Adherent Mammalian Cells Using Laser Ablation Electrospray Ionization Mass Spectrometry in Transmission Geometry.

Most cultured cells used for biomedical research are cultured adherently, and the requisite detachment prior to biochemical analysis might induce chemical changes. This is especially crucial if accurate metabolic measurements are desired, given the rapid turnover of metabolites in living organisms. There are only a few methods available for the nontargeted in situ analysis of small adherent cell populations. Here we show that laser ablation electrospray ionization (LAESI) mass spectrometry (MS) can be used to analyze adherent cells directly, while still attached to the culture surface. To reduce the size of the analyzed cell population, the spot size constraints of conventional focusing in reflection geometry (rg) LAESI had to be eliminated. By introducing transmission geometry (tg) LAESI and incorporating an objective with a high numerical aperture, spot sizes of 10-20 μm were readily achieved. As few as five adherent cells could be specifically selected for analysis in their culturing environment. The importance of in situ analysis was highlighted by comparing the metabolite composition of adherent versus suspended cells. For example, we observed that cells analyzed adherently yielded higher values for the adenylate energy charge (0.90 ± 0.09 for adherent cells vs 0.09 ± 0.03 for suspended cells). Additionally, due to the smaller focal spot size, tg-LAESI enabled the analysis of ∼20 times smaller cell populations compared to rg-LAESI.

Peripheral Blood Mononuclear Cells Enhance Cartilage Repair in in vivo Osteochondral Defect Model.

This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC’s were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.

Mesenchymal niche: the sensor and effector of leukemogenesis.

Mesenchymal stromal cells (MSCs) are nonhematopoietic adherent cell populations in the bone marrow (BM) that exhibit multilineage differentiation potential towards diverse types of tissues. These MSCs are widely used for cell-therapy applications to stimulate injured tissues regeneration. The therapeutic effects of MSCs are primarily exerted through their paracrine secretion of various factors that can stimulate the regenerative potential of endogenous stem cells, promote angiogenesis, and inhibit apoptosis [1]. While ex-vivo expanded MSCs have been widely used for cell-therapy trials, the in vivo identity of these MSCs has been suggested to be a subset of pericytes, and they were demonstrated to be able to reconstitute both endosteal and the perivascular in heterologous implantation studies [2]. While geographically distinct, the perivascular and endosteal in BM exhibit both distinct and common characteristics in cellular entities and cross-talk molecules, comprising primarily of MSCs, endothelial cells, and some cells with neuronal origin [3]. Moreover, recent studies have further identified complex heterogeneity in the cellular entity of the mesenchymal niche. For example, studies have identified early-stage osteoblastic cells expressing runt-related transcription factor 2 (runx2), subsets of MSCs in perivascular regions expressing nestin and leptin-receptor, and primitive (prx-1+) mesenchymal cells expressing C-X-C motif chemokine 12 (CXCL-12) as key functional cells in the niche. These cells express cross-talk molecules such as Jagged-1, CXCL-12, and angiopoietin-1 in order to interact with hematopoietic stem cells (HSCs). Thus, fine orchestration of this microenvironmental cross-talk exerts a key influence on HSCs, controlling their self-renewal, quiescence, and mobilization. While accumulating studies have established the functional impact of the mesenchymal cell in regulating normal HSCs, emerging interest is now focusing on its significance for leukemia stem cells (LSCs), the malignant counterpart of normal HSCs. The key question being addressed is whether the is affected under conditions and, in turn, whether the executes any functions in the leukemogenic process itself. The possible involvement of the in leukemogenesis was first suggested by the observation that LSCs, when transplanted into mice, engraft in BM and competes with normal HSCs [4]. Moreover, leukemia cells transplanted into mice create an abnormal in BM to usurp the transplanted normal HSCs into a tumor [5], suggesting the functional impact of leukemia cells on the niche. Recently, studies have shown that leukemia cells can alter the condition of the mesenchymal in an animal leukemia model, i.e., a transgenic model of chronic myeloid leukemia exhibits defective homing and retention of HSCs in the because of decreased CXCL12 in BM MSCs [6]. Similarly, studies on bcr-abl-transformed leukemia revealed alterations of MSCs and osteoblastic cells during the leukemogenesis [7]. Thus, it appears that the mesenchymal is certainly influenced, under the leukemogenic conditions, to bring about new concept of the leukemic niche that is established by alteration of the normal mesenchymal niche. On the other hand, several experimental studies also raised the possibility that the mesenchymal itself contributes to the leukemogenic process. For example, a higher incidence of myeloproliferative disease was observed in mice when the BM stroma was disrupted with retinoic acid-γ [8] or retinoblastoma [9]. Although these animal models have not clarified a molecular basis for developing and/or expanding the cells, these observations, at least, present the possibility that the microenvironment could also be a driving force in the leukemogenic process. Of note, while most insight was obtained from a series of animal leukemia models, recent studies by our group and others revealed that clinical models of human leukemia exhibit similar alterations in the mesenchymal [10]. This study shows that the mesenchymal in patients undergoes extensive functional and transcriptomic alteration accompanied by the alteration in cross-talk molecules. The altered had a functional impact on the BM microenvironment, leading to selective expansion of leukemia cells while suppressing normal HSCs. Thus, the emerging insights into the indicate that leukemia cells reprogram the mesenchymal to reinforce their own leukemogenesis, leading to the clonal dominance of cells over the normal hematopoietic process. Interestingly, the study by Kim et al. [10] recently demonstrated that the heterogeneity in microenvironmental remodeling can be a parameter for heterogeneous clinical courses in acute myeloid leukemia patients, suggesting that the stromal patterns in leukemia BM can serve as a predictor of prognosis. Based on these collective insights, it appears that MSCs are the responding cells being targeted under the conditions, but also take up a role as active effectors in the leukemogenic process. Accordingly, the mesenchymal represents an attractive target for therapy against various hematological diseases. Microenvironmentbased therapeutic planning will broaden the horizon for more personalized, biological approaches to efficient leukemia treatment by targeting the specific leukemogenic process in the niche.

Novel Evidence That Murine and Human Mesenchymal Stromal Cells Express Functional Gonadotropic Hormone Receptors, Demonstrating the Involvement of the Pituitary gonadotropin–bone Marrow Axis in Hematopoiesis

Background: Mesenchymal stromal cells (MSCs) play an important role in bone marrow (BM) by providing a supportive microenvironment for hematopoietic stem/progenitor cells (HSPCs). MSCs are also employed in organ regeneration as a rich source of several paracrine signals that inhibit apoptosis and promote angiogenesis in damaged tissues. As reported in the literature, several mediators, including a growth factor (HGF), a chemokine (SDF-1), bioactive lipids (S1P, C1P), and extracellular nucleotides (ATP, UTP), affect MSC biology and migration. In parallel, evidence has accumulated that the most primitive mesodermal precursors of MSCs (small BM-residing and peripheral blood (PB)-circulating Sca-1+Lin–CD45– cells in mice and CD133+Lin–CD45– cells in humans) express certain embryonic stem cell markers, such as the transcription factor Oct-4 and the SSEA-1/4 antigens (Stem Cells Dev. 2014;23:689-701), and also express several genes characteristic of migrating primordial germ cells (Leukemia 2010; 24:1450–1461).Hypothesis: Pursuing observations that most primitive human and murine precursors of MSCs express several germline markers, we became interested in whether murine and human MSCs also express gonadotropic hormone receptors, such as receptors for follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL), and whether these receptors are functional.Materials and Methods: Murine and human MSCs were expanded from a population of adherent murine BM or human umbilical cord blood cells, and cells with low passage numbers were employed for analysis. The expression of gonadotropic receptors (FSH-R, LH-R, and PRL-R) was evaluated by RT-PCR, and the functionality of these receptors was tested in assays for proliferation, chemotaxis, adhesion, and phosphorylation of MAPKp42/44 and AKT. In addition, we also evaluated the expression of gonadotropin receptors by purified murine SKL cells and human CD34+ cells.Results. We report here for the first time that both human and murine MSCs and HSPCs express functional gonadotropin receptors. We found that FSH strongly enhanced proliferation of MSCs in vitro as well as expanded the number of these cells in murine BM after prolonged administration in vivo. We found that all these hormones stimulated chemotaxis and adhesion of murine and human MSCs. These functional responses were correlated with phosphorylation of MAPKp42/44 and AKT. At the same time, we observed that pituitary gonadotropin receptors are expressed by murine and human HSPCs and that these hormones stimulate proliferation and expansion of these cells in vivo in BM as well as in clonogeneic assays in vitro if added along with suboptimal doses of colony-stimulating growth factors. Of note, we did not observe significant differences in the effects of FSH, LH, and PRL between male and female cells.Conclusions. We provide for the first time evidence for the existence of a functional pituitary gonadotropin–hematopoiesis signaling axis, which has important implications for hematopoiesis in young individuals, and we will present gene-array data on changes in gene expression in MSCs after stimulation with gonadotropins. Moreover, since the levels of FSH and LH increase in response to a decrease in gonadal function with advanced age, elevated levels of FSH and LH may affect hematopoiesis and may be factors contributing to the development of leukemia. The stimulatory effect of pituitary gonadotropins on MSCs and HSPCs could also be exploited in the clinic in selected cases as a means to enhance hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells.

The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor.

Abstract 1936: The ETS factor ESE3/EHF controls Lin28A and Lin28B and acts as a barrier for stemness in prostate epithelial cells

Abstract Prostate cancer (PCa) is a leading cause of cancer death worldwide. Several studies have provided evidence for the presence of self-renewing cancer stem-like cells (CSCs) in many solid tumors, including prostate cancer. Understanding the molecular mechanism leading to induction of epithelial-to-mesenchymal transition (EMT) and stemness will help to find new therapies for metastatic PCa. We have reported that the ETS factor ESE3/EHF is a tumor suppressor involved in the maintenance of the differentiation state of normal prostate epithelial cells (PrECs). Prostate epithelial cells upon knockdown of ESE3/EHF (ESE3KD PrECs) undergo transformation involving EMT, enlargement of the stem cell compartment with broad reprogramming of the transcriptome. In the process to identify the genes responsible for the activation of stemness in ESE3KD PrECs, we found a significant increase of Lin28A and Lin28B compared to control PrECs. Consistent with their crucial role in stemness maintenance, higher level of Lin28A and Lin28B were detected in the cancer stem cell compartment compared to the bulk population of adherent growing cells and in xenografts derived from the cancer stem cell-enriched population. To relate these effects directly to ESE3/EHF we searched for ETS-binding sites (EBS) in the promoters of the Lin28A and Lin28B. Computational analysis revealed putative EBS in the promoter region of both genes and chromatin immunoprecipitation confirmed the binding of ESE3/EHF to the predicted sites in PrECs and LNCaP cells. Furthermore, ESE3/EHF repressed Lin28A and Lin28B promoter activity in luciferase reporters. Transcriptional repression of Lin28A and Lin28B by ESE3/EHF was confirmed by the enrichment of repressive markers H3K9me and H3K27me in their promoters in LNCAP cells. Knockdown of Lin28A and Lin28B in ESE3KD PrECs by siRNAs reversed anchorage independent growth and in vitro prostatosphere formation. Furthermore, inhibition of Lin28A and Lin28B significantly reduced tumor initiating properties of ESE3KD PrECs in vivo. In contrast, re-expression of Lin28A and Lin28B promoted anchorage-independent growth and increased prostatosphere formation and self renewal in PrECs and LNCaP cells. Together, these data indicate that ESE3/EHF maintains in a repressed state Lin28A and Lin28B and constitutes a barrier to dedifferentiation of prostate epithelial cells. These data indicate also a role of Lin28A and Lin28B in the expansion and maintenance of the stem cell compartment in prostate cancer and underscore their validity as potential therapeutic targets. Citation Format: Domenico Albino, Gianluca Civenni, Mahnaz Nikpour, Carlo V. Catapano, Giuseppina M. R. Carbone. The ETS factor ESE3/EHF controls Lin28A and Lin28B and acts as a barrier for stemness in prostate epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1936. doi:10.1158/1538-7445.AM2014-1936