Stem Cell Growth Factor Research Articles

Abstract 4695: Targeting the LGR5 complex with BNC101 to improve checkpoint inhibitor therapy in colorectal cancer

Abstract LGR5 is a well characterised marker of intestinal stem cells found at the base of intestinal crypts and a receptor for R-spondins, potent Wnt signalling modulators and stem cell growth factors. Overexpression of LGR5 in colorectal tumor cells has been shown to be a predicative marker of higher relapse rates in CRC patients. BNC101 is a first-in-class high affinity anti-LGR5 humanized monoclonal antibody currently in a Phase I clinical trial in patients with recurrent metastatic CRC. BNC101 has been shown pre-clinically to have anti-tumor activity in multiple CRC patient derived xenografts and limiting dilution re-implantation assays consistent with the hypothesis that LGR5 is a functional cancer stem cell (CSC) target in CRC. Recent evidence suggests that CSCs or treatment resistant cells, due to distinct gene and antigen expression profiles, may share the same immune privilege that is afforded to normal stem cells. Immunologic therapies with checkpoint inhibitors directed against whole tumors are largely biased toward differentiated tumor cells which form the bulk of the tumor. CSCs may also have an immunosuppressive phenotype, allowing them to evade and dampen the anti-tumor host immune response. The syngeneic murine MC38 colorectal cancer model was used to explore the potential synergies of BNC101 and anti-PD-1 treatments. The combination of murine versions of both antibodies drove a reduction (35%) in tumoral Tregs (FoxP3+) when BNC101 and anti-PD-1 were used compared to anti-PD-1 treatment alone. This is a significant finding and shows early efficacy in keeping with the notion that CSCs create an immunosuppressive environment which can be targeted in combination for a better immunotherapeutic response. The activation of antibody-dependent cell-mediated cytotoxicity (ADCC) was demonstrated using BNC101 treatment of cells. Membrane dye tracked U-937 monocytes (which express FcR) were able to specifically cross bind with CHO-LGR5 cells but only in the presence of BNC101 (an IgG1). This potentially indicates a further adaptive immune priming mechanism with the ability to complement the action of checkpoint inhibitors. Understanding the role of CSCs in tumor immunity will help guide future clinical applications of immune checkpoint inhibitors in CSC-driven solid tumors. In addition, combination of immune checkpoint therapies with CSC targeting agents may improve the clinical utility of each approach. These findings support clinical evaluation of BNC101 in combination with checkpoint inhibitors. Releasing the immunosuppressive capabilities of CSC with BNC101 driven targeting of the tumor may extend the reach of the immune system, whereby checkpoint inhibitors are able to leverage greater therapeutic benefit to a larger patient population and extend duration of response. Citation Format: Daniel J. Inglis, Donna M. Beaumont, Tina C. Lavranos. Targeting the LGR5 complex with BNC101 to improve checkpoint inhibitor therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4695. doi:10.1158/1538-7445.AM2017-4695

Abstract 1098: Delta-tocotrienol chemosensitizes human pancreatic tumor metastasis to gemcitabine targeting cancer stem cells

Abstract Background: Pancreatic cancer, a lethal malignancy is the fourth leading cause of cancer-related deaths in the United States. The standard chemotherapy with gemcitabine is ineffective in patients with metastatic tumors. Pancreatic cancer stem cells (CSCs) have been implicated in the development of pancreatic cancer metastasis, resistance to chemotherapy and its recurrence following surgical extirpation. We have shown that natural vitamin E δ-tocotrienol is the most bioactive tocotrienols against pancreatic cancer in vitro as well as in vivo models. The purpose of this study was to evaluate the chemosensitization of pancreatic tumor metastasis by δ-tocotrienol to gemcitabine in vitro as well as in vivo. Methods: In vitro human metastatic pancreatic cancer cells L3.6pl and pancreatic cancer stem cells (PCSC) were treated with gemcitabine (5 μM) and δ-tocotrienol (50 μM) alone and in combination. Treated cells used for epithelial to mesenchymal transition (EMT), migration/invasion, microsphere/spheroid, and signaling markers assays. PCSC expressing luciferase was orthotopically implanted into pancreas of Athymic nude mice (n = 20) and after one week they were randomized into four groups: 1) vehicle control (ethanol extracted olive oil), 2) gemcitabine (100 mg/kg, IP, twice a week), 3) δ-tocotrienol (200 mg/kg, orally twice a day) and 4) gemcitabine + δ-tocotrienol. The treatment was continued for 4 weeks. The tumor volume, tumor weight, metastasis were recorded. Results: Gemcitabine slightly inhibited the growth, migration/invasion of L3.6pl and PCSC in vitro and PCSC tumor growth (tumor volume and weight) in vivo. δ-tocotrienol significantly inhibited the growth, microsphere/spheroid, migration/invasion, EMT(E-cadherin to vimentin), angiogenesis (VEGF), PCSC transcription factors (Nanog, Oct4, and Sox2), tumor growth and liver/lung metastasis compared to control. The combination of both drugs synergistically inhibited the cancer and stem cell growth, EMT, migration/invasion, microsphere/spheroid, metastasis, angiogenesis, and PCSC transcription factors in vitro as well as in vivo. Conclusion: Vitamin E δ-tocotrienol chemosensitizes human pancreatic tumor metastasis to gemcitabine through inhibition of EMT, migration, invasion, angiogenesis, cancer stem cell self-renewal, tumor growth and metastasis. Citation Format: Kazim Husain, Said M. Sebti, Mokenge P. Malafa. Delta-tocotrienol chemosensitizes human pancreatic tumor metastasis to gemcitabine targeting cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1098. doi:10.1158/1538-7445.AM2017-1098

205 - The stem cell growth factor receptor KIT is not expressed on interstitial cells in bladder

205 - The stem cell growth factor receptor KIT is not expressed on interstitial cells in bladder

Plasma Cytokines Correlated With Disease Characteristics, Progression-Free Survival, and Overall Survival in Testicular Germ-Cell TumorPatients.

Cytokines are the communicators of immune system and are involved in all immune responses. The aim of this study was to assess the correlation among plasma cytokines, patient and tumor characteristics, and clinical outcome in chemonaive testicular germ-cell tumor (TGCT) patients. This study included 92 metastatic chemotherapy-naive TGCT patients treated with platinum-based chemotherapy from July 2010 to March 2014. Plasma was isolated before first administration of chemotherapy, and the concentration of 51 plasma cytokines were analyzed using multiplex bead arrays. At a median follow-up of 33.2 months (range, 0.1-54.8 months), 10.9% of patients experienced disease progression, and 7.6% died. Several cytokines were associated with different baseline clinicopathologic features. Elevated plasma levels of interferon (IFN)-α2, interleukin (IL)-2Rα, IL-16, hepatocyte growth factor (HGF), and monocyte chemotactic protein (MCP)-3 were significantly associated with worse progression-free survival and overall survival (OS). Moreover, elevated levels of stem-cell growth factor (SCGF)-β were also associated with worse OS. Patients with elevated levels of all 6 cytokines experienced significantly worse outcomes compared to patients who had fewer than 6 cytokines elevated (hazard ratio= 12.06; 95% confidence interval, 7.39-19.49; P= .002 for progression-free survival, and hazard ratio= 39.65; 95% confidence interval, 25.03-62.18; P< .00001 for OS, respectively). Results were independent of International Germ Cell Cancer Collaborative Group criteria. We found a correlation among progression free-survival, OS, and circulating cytokines in TGCT. This suggests the existence an association between plasma cytokines and baseline clinicopathologic features in TGCT. Plasma cytokines could be used for identification of high-risk patients who are candidates for new therapeutic approaches.

Adenovirus-Mediated Expression of BMP-2 and BFGF in Bone Marrow Mesenchymal Stem Cells Combined with Demineralized Bone Matrix For Repair of Femoral Head Osteonecrosis in Beagle Dogs

Background: This study investigated the effect of using adenovirus-mediated expression of bone morphogenetic protein 2 (Ad-BMP-2) and basic fibroblast growth factor (bFGF) in bone marrow mesenchymal stem cells (BMSCs) in combination with a demineralized bone matrix (DBM) to repair osteonecrosis of the femoral head (ONFH) in Beagle dogs. Methods: A total of 30 Beagle dogs were selected for the isolation of BMSCs, which were cultured and transfected with the recombinant adenovirus vector Ad-BMP2-bFGF-GFP (carrying BMP-2 and bFGF) or a control adenovirus plasmid (encoding green fluorescent protein (Ad-GFP)). The expression of the transfected BMP-2 and bFGF proteins was detected by Western blotting. After transfection, the BMSCs were induced to undergo osteoblastic differentiation. The DBM was prepared to construct a DBM/BMSC complex. Beagle models of canine femoral head defects and necrosis were established and divided into control, DBM, DBM/BMSC, vector Ad-BMP2-bFGF-GFP and Ad-GFP groups. The composite graft was then implanted, and new bone morphology was visualized via X-ray at 3, 6 and 12 weeks after the operation. Hematoxylin and eosin (HE) staining and Masson’s trichrome staining were used to identify new bone formation. Immunohistochemistry was performed to calculate the density of new blood vessels. The compressive and bending strength of the BMSCs was evaluated at 12 weeks after the operation. Results: BMSCs were successfully isolated. The protein expression of BMP-2 and bFGF was significantly higher in the Ad-BMP-2/bFGF group than the normal and Ad-GFP groups. Compared with the control group, at 12 weeks after the operation, the DBM, DBM/BMSC, vector Ad-BMP2-bFGF-GFP and Ad-GFP groups showed a larger area of new bone, higher X-ray scores, greater neovascularization density, and increased compressive and bending strength. The most significant modifications occurred in thevector Ad-BMP2-bFGF-GFP group. Conclusion: The results indicate that the use of Ad-BMP-2/bFGF-modified BMSCs in conjunction with DBM could successfully repair ONFH in a dog model by promoting bone formation and angiogenesis.

The stem cell growth factor receptor KIT is not expressed on interstitial cells in bladder.

The mast/stem cell growth factor receptor KIT has long been assumed to be a specific marker for interstitial cells of Cajal (ICC) in the bladder, with possible druggable perspectives. However, several authors have challenged the presence of KIT + ICC in recent years. The aim of this study was therefore to attempt to clarify the conflicting reports on KIT expression in the bladder of human beings, rat, mouse and guinea pig and to elucidate the possible role of antibody‐related issues and interspecies differences in this matter. Fresh samples were obtained from human, rat, mouse and guinea pig cystectomies and processed for single/double immunohistochemistry/immunofluorescence. Specific antibodies against KIT, mast cell tryptase (MCT), anoctamin‐1 (ANO1) and vimentin were used to characterize the cell types expressing KIT. Gut (jejunum) tissue was used as an external antibody control. Our results revealed KIT expression on mast cells but not on ICC in human, rat, mouse and guinea pig bladder. Parallel immunohistochemistry showed KIT expression on ICC in human, rat, mouse and guinea pig gut, which confirmed the selectivity of the KIT antibody clones. In conclusion, we have shown that KIT + cells in human, rat, mouse and guinea pig bladder are mast cells and not ICC. The present report is important as it opposes the idea that KIT + ICC are present in bladder. In this perspective, functional concepts of KIT + ICC being involved in sensory and/or motor aspects of bladder physiology should be revised.

Alteration of the Hematopoietic Components, Mesenchymal Stromal Cells and Vascular Elements in the Bone Marrow Niche in Myelodysplastic Syndrome: Refractory Cytopenia with Multilineage Dysplasia

Introduction:The myelodysplastic syndromes (MDS) are clonal disorders characterized by cytopenias and abnormal hematopoiesis. Though there are reports of perturbations in the hematopoietic stem cells (HSC) and the mesenchymal stromal cells (MSC) as well as other elements of the bone marrow (BM) niche in an instructive or permissive manner leading to the genesis of ineffective hematopoiesis in this condition, most studies have evaluated single elements. Here we demonstrate altered HSC, MSC and the vascular niche elements in patients with MDS - Refractory cytopenia with multilineage dysplasia (RCMD).Methods and results: Bone marrow aspirates from patients with RCMD (n=12, mean age 54.33±14.51 years) were compared with those from age-matched controls (n=18, mean age 47.78±18.60 years) who had a ’normal’ marrow obtained for other diagnostic purposes. Cytogenetic analysis showed abnormal karyotypes in seven patients and normal karyotypes in five. The abnormalities seen were as follows: deletion 7q/monosomy 7 (four patients), deletion 5q (three patients), loss of Y (two patients), monosomy 5, deletion 20q (one each), complex karyotypes (four patients). Phenotypic enumeration of HSPCs revealed a marked decrease in the frequency of highly purified HSCs (Lin-CD34+CD38-CD90+CD45RA-) in RCMD (0.04135±0.01748%, n=11) when compared to controls (0.3168±0.05266%, n=13, p<0.0001) (Figure 1). Patients with RCMD also had increased common myeloid progenitors (CMP) (3.221 ± 0.7478%, n=8) compared to control (1.243±0.4463%, n=10, p<0.0302) and loss of granulocyte-macrophage progenitors (GMP) (0.4863±0.1638%, n=8) compared to controls (2.047±0.5422%, n=10, p<0.0242) (Figure 2).Assessment of the frequency of de novo MSCs (CD31-CD45/CD71- population) expressing CD271 and/or CD146, indicating the more primitive population, in total nucleated cells showed increased CD271+CD146-MSCs (0.632±0.2, n=5) compared to controls (0.2000±0.05158, n=6, p<0.0401) as also the CD271+CD146+ MSCs (0.2900±0.09803, n=5) in RCMD patients when compared to controls (0.02861±0.01354, n=6, p<0.0172) (Figure 3). We also evaluated in vitro cultured MSCs (P4) in these patients. CD271+CD146+ MSCs within total cultured MSCs were higher (0.1900±0.06429%, n=3) than in controls (0.0040±0.0040%, n=3, p<0.0447). RCMD MSCs had significantly lower proliferation index (32±3.7%, n=3) compared to controls (60±9.2%, n=3, p<0.0479). Cell cycle analysis of MSC showed significantly lower numbers in G0 in RCMD (0.1567 ± 0.07881 %, n=3) compared to control (0.6400 ± 0.1007 %, n=3; p<0.0194). Apoptosis was much higher in RCMD MSCs (2.700±0.8007%, n=3) compared to controls (0.03633±0.01802%, n=3, P<0.0292). No significant differences in expression profile of stem cell maintenance related cytokines and growth factors (CXCL12a, SCF, VEGF, ANGPT and LIF ) or components of Notch (Notch1, Notch3, Jagged-1, Delta like-1 and Hes1) and Wnt (Dkk1 and Dkk-2) pathways were found between RCMD and control MSCs within the limited numbers evaluated. Interestingly, unlike controls, immunofluorescence imaging of bone marrow trephine from MDS-RCMD revealed CD271+CD146+ MSCs co-localized with sinusoids and in direct contact with CD34+ (<5% blast) HSC/progenitor cells (HSPCs).Conclusion: Our data shows that in patients with MDS-RCMD, both qualitative and quantitative abnormalities exist in the HSC, HSPC and niche elements. We also show that the cytopenia could be related to decreased numbers of primitive HSCs and a differentiation arrest at the CMP stage. Primitive MSCs are reduced, and those that exist show poorer proliferative and survival features. Further studies are needed to understand the cause and effect relationship of these changes. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of small molecule tyrosine kinase inhibitors and cytotoxic drugs.

Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 μM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2). Finally, of the TKIs, only treatment with afatinib, neratinib and dasatinib were able to reduce the migration of HER-2 overexpressing SKOV3 cells. We did not find any significant association between the expression of putative ovarian CSC marker, HER family members, c-MET, ALK, and IGF-IR and the response to the irreversible HER TKIs. Our results support the need for further investigations of the therapeutic potential of these irreversible HER family blockers in ovarian cancer, and the therapeutic potential of dasatinib when used in combination with the inhibitors of the HER family members in ovarian cancer.

Abstract IA04: Lgr5 stem cell-based organoids in human disease

Abstract The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost ‘generic’ marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear tongue and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so called ‘mini-guts’ in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D cultures systems have been developed for the Lgr5+ve stem cells of human stomach, liver, pancreas, prostate and kidney. Using CRISPR/Cas9 technology, genes can be efficiently modified in organoids of various origins. Citation Format: Hans Clevers. Lgr5 stem cell-based organoids in human disease. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA04.

Stem cell growth factor receptor in canine vs. feline osteosarcomas.

Osteosarcoma is considered the most common bone cancer in cats and dogs, with cats having a much better prognosis than dogs, since the great majority of dogs with osteosarcoma develop distant metastases. In search of a factor possibly contributing to this disparity, the stem cell growth factor receptor KIT was targeted, and the messenger (m)RNA and protein expression levels of KIT were compared in canine vs. feline osteosarcomas, as well as in normal bone. The mRNA expression of KIT was quantified by reverse transcription-quantitative polymerase chain reaction, and was observed to be significantly higher in canine (n=14) than in feline (n=5) osteosarcoma samples (P<0.001). KIT protein expression was evaluated by immunohistochemistry, which revealed that 21% of canine osteosarcoma samples did not exhibit KIT staining in their neoplastic cells, while in 14% of samples, a score of 1 (<10% positive tumour cells) was observed, and in 50% and 14% of samples, a score of 2 (10–50% positivity) and 3 (>50% positivity), respectively, was observed. By contrast, the cancer cells of all the feline bone tumour samples analysed were entirely negative for KIT. Notably, canine and feline osteocytes of healthy bone tissue lacked any KIT expression. These results could be the first evidence that KIT may be involved in the higher aggressiveness of canine osteosarcoma compared with feline osteosarcoma.

Fractones: extracellular matrix niche controlling stem cell fate and growth factor activity in the brain in health and disease.

The stem cell niche refers to a specific microenvironment where stem cells proliferate and differentiate to produce new specialized cells throughout an organism's adulthood. Growth factors are crucial signaling molecules that diffuse through the extracellular space, reach the stem cell niche, and ultimately promote stem cell proliferation and differentiation. However, it is not well known how multiple growth factors, often with antagonistic activities, work together in the stem cell niche to select target stem cell populations and determine stem cell fate. There is accumulating evidence suggesting that extracellular matrix (ECM) molecules play an important role in promoting growth factor access and activity in the stem cell niche. In the adult brain neurogenic zone, where neural stem cells (NSCs) reside, there exist specialized ECM structures, which we have named fractones. The processes of NSC allow them to come into contact with fractones and interact with its individual components, which include heparan sulfate proteoglycans (HSPGs) and laminins. We have demonstrated that fractone-associated HSPGs bind growth factors and regulate NSC proliferation in the neurogenic zone. Moreover, emerging results show that fractones are structurally altered in animal models with autism and adult hydrocephalus, as demonstrated by changes in fractone size, quantity, or HSPG content. Interestingly, ECM structures similar to fractones have been found throughout β-amyloid plaques in the brain of patients with Alzheimer's disease. Pathological fractones may cause imbalances in growth factor activity and impair neurogenesis, leading to inflammation and disorder. Generally speaking, these stem cell niche structures play a potentially vital role in controlling growth factor activity during both health and disease.

Molecular subtypes of gastrointestinal stromal tumor requiring specific treatments

This article reviews and discusses the current literature on how molecular subtyping of gastrointestinal stromal tumor (GIST) impacts decision-making in clinical practice. Genotyping has not yet been used for prognostication of localized GIST. Recent findings indicate that mutations in platelet-derived growth factor receptor alpha (PDGFRA) and stem cell growth factor receptor (KIT) exon 11 duplication mutations are associated with a favorable prognosis. Subgroup analyses of randomized trials provide first evidence on how genotyping predicts clinical benefit in the adjuvant setting. In the palliative setting, genotyping should be performed for dose selection but may also be relevant in patients who are intolerant to imatinib. For patients whose tumors harbor the notoriously resistant D842V PDGFRA mutations, novel inhibitors have entered clinical trials, which may end the therapeutic deadlock. Several novel targets have been identified that are currently being investigated in GIST, including inhibitors against fibroblast growth factor receptor, hepatocyte growth factor receptor, mitogen-activated protein, and phosphoinositide 3-kinase. Preliminary clinical data suggest that predictive markers will be needed to define sensitive molecular subgroups. Genotyping should be an integral part of clinical management of GIST as it aids in prognostication, prediction of efficacy, and selection of drugs and dosing. Genotyping reduces the risk of both unnecessary adjuvant treatment as well as undertreatment and overtreatment in the palliative setting.

A novel method to limit breast cancer stem cells in states of quiescence, proliferation or differentiation: Use of gel stress in combination with stem cell growth factors.

The majority of cancer stem cells exist in the G0, or quiescent phase of the cell cycle. However, the cells can escape quiescence following routine radiotherapy and chemotherapy, resulting in tumor recurrence. Presently, achieving the accurate regulation of cancer stem cell growth in order to study a specific state, including the quiescent (mostly G0 or G1 phase), proliferative (mostly S phase) or differential (mostly G2/M phase) states, can be challenging. This makes the determination of cell cycle state-specific characteristics and analysis of potential intervention treatments difficult, particularly for quiescent cells. Breast cancer stem cells were cultured on a soft or hard agar matrix surface in the presence or absence of stem cell growth factors. Cells could be successfully limited in either the quiescent, proliferative or differentiated states. These findings provide a foundation for further study of the cell cycle in breast cancer stem cells.

Small cell lung cancer: Circulating tumor cells of extended stage patients express a mesenchymal-epithelial transition phenotype

ABSTRACTSmall cell lung cancer (SCLC) is distinguished by aggressive growth, early dissemination and a poor prognosis at advanced stage. The remarkably high count of circulating tumor cells (CTCs) of SCLC allowed for the establishment of permanent CTC cultures at our institution for the first time. CTCs are assumed to have characteristics of cancer stem cells (CSCs) and an epithelial-mesenchymal transition (EMT) phenotype, but extravasation of tumors at distal sites is marked by epithelial features. Two SCLC CTC cell lines, namely BHGc7 and BHGc10, as well as SCLC cell lines derived from primary tumors and metastases were analyzed for the expression of pluripotent stem cell markers and growth factors. Expression of E-cadherin and β-Catenin were determined by flow cytometry. Stem cell-associated markers SOX17, α-fetoprotein, OCT-3/4, KDR, Otx2, GATA-4, Nanog, HCG, TP63 and Goosecoid were not expressed in the 2 CTC lines. In contrast, high expression was found for HNF-3β/FOXA2, SOX2, PDX-1/IPF1 and E-cadherin. E-cadherin expression was restricted to the 2 CTCs and 2 cell lines derived from pleural effusion (SCLC26A) and bone metastases (NCI-H526), respectively. Thus, these SCLC CTCs established from extended disease SCLC patients lack expression of stem cell markers which suppress the epithelial phenotype. Instead they express high levels of E-cadherin consistent with a mesenchymal-epithelial transition (MET or EMrT) and form large tumorospheres possibly in response to the selection pressure of first-line chemotherapy. HNF-3β/FOXA2 and PDX-1/IPF1 expression seem to be related to growth factor dependence on insulin/IGF-1 receptors and IGF-binding proteins.

Abstract IA12: Lgr5 stem cell-based organoids in human disease

Abstract The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost ‘generic’ marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear tongue and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so called ‘mini-guts’ in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D cultures systems have been developed for the Lgr5+ve stem cells of human stomach, liver, pancreas, prostate and kidney. Using CRISPR/Cas9 technology, genes can be efficiently modified in organoids of various origins. Citation Format: Hans Clevers. Lgr5 stem cell-based organoids in human disease. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA12.

Maintenance of Proliferation and Adipogenic Differentiation by Fibroblast Growth Factor-2 and Dexamethasone Through Expression of Hepatocyte Growth Factor in Bone Marrow-derived Mesenchymal Stem Cells

Several studies have investigated the various effects of dexamethasone (Dex) on the proliferation and differentiation of mesenchymal stem cells (MSCs). Previously, we reported that co-treatment with L-ascorbic acid 2-phosphate and fibroblast growth factor (FGF)-2 maintained differentiation potential in MSCs through expression of hepatocyte growth factor (HGF). In this study, we investigated the effects of co-treatment with FGF-2 and Dex on the proliferation and differentiation potential of MSCs during a 2-month culture period. Co-treatment with FGF-2 and Dex increased approximately a 4.7-fold higher accumulation rate of MSC numbers than that by FGF-2 single treatment during a 2-month culture period. Interestingly, co-treatment with FGF-2 and Dex increased expression of HGF and maintained adipogenic differentiation potential during this culture period. These results suggest that co-treatment with FGF-2 and Dex preserves the proliferation and differentiation potential during long-term culture.

Changing Paradigms in Cranio-Facial Regeneration: Current and New Strategies for the Activation of Endogenous Stem Cells.

Craniofacial area represent a unique district of human body characterized by a very high complexity of tissues, innervation and vascularization, and being deputed to many fundamental function such as eating, speech, expression of emotions, delivery of sensations such as taste, sight, and earing. For this reasons, tissue loss in this area following trauma or for example oncologic resection, have a tremendous impact on patients' quality of life. In the last 20 years regenerative medicine has emerged as one of the most promising approach to solve problem related to trauma, tissue loss, organ failure etc. One of the most powerful tools to be used for tissue regeneration is represented by stem cells, which have been successfully implanted in different tissue/organs with exciting results. Nevertheless, both autologous and allogeneic stem cell transplantation raise many practical and ethical concerns that make this approach very difficult to apply in clinical practice. For this reason different cell free approaches have been developed aiming to the mobilization, recruitment, and activation of endogenous stem cells into the injury site avoiding exogenous cells implant but instead stimulating patients' own stem cells to repair the lesion. To this aim many strategies have been used including functionalized bioscaffold, controlled release of stem cell chemoattractants, growth factors, BMPs, Platelet–Rich-Plasma, and other new strategies such as ultrasound wave and laser are just being proposed. Here we review all the current and new strategies used for activation and mobilization of endogenous stem cells in the regeneration of craniofacial tissue.

Effects of Panax notoginseng saponins on proliferation and differentiation of rat embryonic cortical neural stem cells

BackgroundWe aimed to study the effect of Panax notoginseng saponins (PNS) on the proliferation, differentiation, self-renewal, and expressions of basic fibroblast growth factor (bFGF) and brain-derived neurotrophic factor (BDNF) in rat embryonic neural stem cells (NSCs). MethodsCortical stem cells were isolated from rat embryos on Embryonic Day 17 (E17) and identified by nestin expression. Subsequently, primary culture, subculturing, and single cell cloning were performed on the cells. After the first cell passage (P1), the cells were resuspended and divided into a control group and a treatment group. Control cells were cultured in serum-free basal culture medium with B27 and dulbecco's modified eagle medium (DMEM)/F12. The same medium supplemented with PNS (100 μg/mL) was used to culture cells in the treatment group. Both groups were incubated at 37°C in a 5% CO2 incubator. Immunocytochemistry was performed 4 days after incubation. ResultsPrimary, P1, and P2 cells in the treatment group formed neurospheres, as did single cell clones of the P1 cells in this group. After being cultured for 4 days, the number of nestin-, proliferating cell nuclear antigen (PCNA)-, Tuj-1-, neurofilament (NF)-, vimentin-, glial fibrillary acidic protein (GFAP)-, bFGF-, and BDNF-positive cells significantly increased in the treatment group in comparison to the control group. All positively stained cells could form clear clusters. ConclusionPNS can promote rat embryonic cortical NSC survival, self-renewal, proliferation, and differentiation through neurotrophic factors by autocrine or paracrine signaling.

The induction and cryopreservation of erythroid progenitor cells derived from umbilical cord blood mononuclear cells

目的探索含多种细胞因子无血清培养基诱导脐血单个核细胞体外分化为红系祖细胞效率及红系祖细胞的保存方法。方法利用羟乙基淀粉沉降脐血中红细胞，人淋巴细胞分离液（Ficoll）分离单个核细胞，采用含FMS样酪氨酸激酶3配体、干细胞生长因子、胰岛素样生长因子1、重组人红细胞生成素的无血清培养基进行体外诱导脐血单个核细胞向红系祖细胞分化，并将诱导的红系祖细胞用不同的冻存液进行冷冻保存，观察红系祖细胞诱导、分化能力和红系祖细胞冷冻保存效果。结果随着诱导时间延长，细胞总数明显增多，培养14 d红系祖细胞扩增约110倍，存活率为（88.92±0.95）%，红系祖细胞特异性标志CD71+细胞占（86.77±9.11）%，CD71 +/CD235a+细胞占（64.47±16.67）%。诱导10 d多为中幼红细胞，可见血红蛋白表达的阳性细胞；诱导14 d开始出现晚幼红细胞，细胞沉淀呈红色。诱导7 d红系集落数为326.00±97.96，高于诱导前（61.60±20.03）。10％二甲基亚砜（DMSO）+2％人血清白蛋白保存细胞复苏后红系祖细胞存活率、回收率分别为（90.32±1.80）%、（93.66±1.87）%，将50％自体脐血浆与10% DMSO、2％人血清白蛋白联用可获得更好的保护效果。结论该无血清培养基可高效诱导、扩增红系祖细胞，10% DMSO+2％人血清白蛋白+50％自体脐血血浆可很好保存红系祖细胞。

Local delivery of stem cell growth factors with injectable hydrogels for myocardial therapy

Event Abstract Back to Event Local delivery of stem cell growth factors with injectable hydrogels for myocardial therapy Renae Waters1, 2, Settimio Pacelli1, Ryan Maloney1, 2 and Arghya Paul1, 2 1 University of Kansas, Chemical and Petroleum Engineering, United States 2 School of Engineering, University of Kansas, Bioengineering Graduate Program, United States Introduction: Stem cell therapy offers a promising approach to regenerate damaged cardiac tissue after acute myocardial infarction. Recent evidences have attributed such improvement to the broad repertoire of angiogenic paracrine factors (growth factors, chemokines and exosomes) secreted by the stem cells, known as secretome[1]. Here, we intend (i) to efficiently bio-manufacture cell secretome from 3D stem cell aggregates using a microfluidic system and (ii) efficiently deliver the harnessed secretome for cardiac repair and revascularization applications using a hydrogel-based injectable delivery system. Experiments and Results: As a first step, a microfluidic device consisting of deep concave microwell arrays was fabricated using soft lithography microscale techniques. Results demonstrate that bone marrow derived human mesenchymal stem cell (hMSC) suspensions, injected to the microfluidic chip through inlet port, form uniformly sized aggregates (~220μm diameter) within 2 days. Computational analysis confirmed rapid oxygen diffusion in the microwells. 3 days post-incubation, hMSC secretome (4.5-9 times increase in angiogenic protein concentrations compared to 2D culture including VEGF, FGF, Angiogenin) was collected, leaving behind the hMSC aggregates at the bottom of the well. As a next step, a physically cross-linked injectable nanocomposite hydrogel, comprising of silicate nanoplatelets and gelatin, was formulated to deliver the harnessed hMSC secretome to the target site. In vitro studies confirmed controlled release of bioactive hMSC growth factors from the hydrogel which induced proliferation of human endothelial cells (HUVEC). For in vivo studies rat model with acute myocardial infarction (n=5) was used. The secretome carrying hydrogel was injected intramyocardially at the per-infarct region of the heart, post infarction as mentioned in earlier work[2]. After 3 weeks, the animals were sacrificed and the injected tissue regions were examined for histological analysis. Masson’s Trichome staining confirmed reduced scar area in the treated Secretome+ group (3D cell aggregates) compared to control group (secretome from 2D cell culture). Immunostaining with CD31 antibodies also confirmed significantly high local myocardial angiogenesis at the injected site in Secretome+ group. These data confirms the angiogenic therapeutic potential of the developed hydrogel in vitro and in vivo. Conclusion: Take together, this study reports a novel strategy to utilize cell secretome, as an alternate to traditional cell therapy, for myocardial regeneration therapy. The strategy can also be used for other biomedical applications, such as wound healing and vascular tissue engineering. Arghya Paul likes to acknowledge the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences, National Institutes of Health (NIH), under Award Number P20GM103638.