Putative Stem Research Articles

Significant co-expression of putative cancer stem cell markers, EpCAM and CD166, correlates with tumor stage and invasive behavior in colorectal cancer

BackgroundThe crucial oncogenic role of cancer stem cells (CSCs) in tumor maintenance, progression, drug resistance, and relapse has been clarified in different cancers, particularly in colorectal cancer (CRC). The current study was conducted to evaluate the co-expression pattern and clinical significance of epithelial cell adhesion molecules (EpCAM) and activated leukocyte cell adhesion (CD166 or ALCAM) in CRC patients.MethodsThis study was carried out on 458 paraffin-embedded CRC specimens by immunohistochemistry on tissue microarray (TMA) slides.ResultsElevated expression of EpCAM and CD166 was observed in 61.5% (246/427) and 40.5% (164/405) of CRC cases. Our analysis showed a significant positive association of EpCAM expression with tumor size (P = 0.02), tumor stage (P = 0.007), tumor differentiate (P = 0.005), vascular (P = 0.01), neural (P = 0.01), and lymph node (P = 0.001) invasion. There were no significant differences between CD166 expression and clinicopathological parameters. Moreover, the combined analysis demonstrated a reciprocal significant correlation between EpCAM and CD166 expression (P = 0.02). Interestingly, there was a significant positive correlation between EpCAM/CD166 phenotypes expression and tumor stage (P = 0.03), tumor differentiation (P = 0.05), neural, and lymph node invasion (P =0.01).ConclusionsThe significant correlation of EpCAM and CD166 expression and their association with tumor progression and aggressive behavior is the reason for the suggestion of these two CSC markers as promising targets to promote novel effective targeted-therapy strategies for cancer treatment in the present study.

Abstract LB131: Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells

Abstract Aim: We investigated the mechanisms driving the concomitant overexpression of ABCG2 and CD133 in cancer stem cells (CSCs) and examined the effect of modulating the underlying machinery on anticancer drug response. Background: Multidrug resistance is a major obstacle to chemotherapy. It is usually associated with increased efflux of anticancer drugs by ATP-binding cassette transporters including ABCG2. ABCG2 is also a key survival factor for CSCs, which lead to recurrence. CSCs have been identified in patient tumor specimens and in cancer cell lines. The CSC hypothesis suggests that anticancer therapies have to target and destroy all residual CSCs in order to produce a durable response. CD133 is a well-characterized stem cell biomarker and it plays crucial role in the self-renewal and tumorigenesis of CSCs. Importantly, ABCG2 and CD133 are co-expressed in CSCs, yet the mechanism(s) driving their concomitant high expression remain elusive. mRNA polyadenylation is a key step regulating the maturation of eukaryotic mRNAs by adding a poly A tail to their 3' ends. Alternative polyadenylation, commonly observed in cancer cells, is a phenomenon responsible for the production of multiple mRNA isoforms bearing different length of 3'-untranslated region (3'UTR). Method: Sub-population of cancer cells from three ABCG2-overexpressing multidrug resistant colon cancer cell lines were sorted by Hoechst dye exclusion or CSC markers (DCLK1+LGR5+BMI1+), respectively, as side population (SP) and putative CSC cells. Expression of ABCG2, CD133, and a few master mRNA polyadenylation trans-acting regulators was compared in SP (or CSC) versus non-SP (or non-CSC) cells. Altered 3'UTR isoforms of ABCG2 and CD133 were examined by 3'RACE assay and 3'UTR isoform specific PCR. The anticancer activity of common chemotherapeutic drugs was examined after genetic modulation of the key mRNA polyadenylation regulators identified. Result: The mRNA stability and expression of both ABCG2 and CD133 were found to be remarkably higher in SP cells (or CSC cells) than in non-SP cells (or non-CSC cells). By 3'RACE assay, shortening of ABCG2 and CD133 3'UTR was evidenced in SP (and CSC) cells but not in non-SP (and non-CSC) cells, thereby increasing mRNA stability and/or translation by escaping the repression from regulatory elements found on long mRNA 3'UTR. Expression of two master mRNA polyadenylation regulators (PABPN1 and CSTF2) was found to be remarkably lower and higher, respectively, in SP (and CSC) than in non-SP (and non-CSC) cells, presumably allowing the preferential utilization of proximal 3'UTR to mediate high expression of ABCG2 and CD133. Genetic silencing of CSTF2 was shown to enhance the anticancer effect of SN-38 and reduce the colony formation capacity of CSC cells. Conclusion: A novel mRNA 3'UTR shortening machinery was shown to mediate the simultaneous overexpression of ABCG2 and CD133 in SP and CSC cells. It may represent useful drug target for circumvention of resistance and eradication of CSCs. Citation Format: Kenneth K.W. To. Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB131.

Abstract 2435: Transcription Factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression

Abstract Ubiquitin-specific peptidase 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in various human cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in a number of human cancers including lung cancer. However, USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown. We set out to characterize and identify potential transcription factors that regulate USP22 expression in human non-small cell lung cancers (NSCLC). A luciferase reporter driven by the promoter region from -5085 to +55 of USP22 gene was constructed to screen a customized siRNA library targeting about 100 selected transcription factors that are expected to bind to the USP22 promoter. Luciferase reporter assays have revealed that knockdown of SP1 and activating transcription factor 3 (ATF3) promote USP22 expression, while knockdown of transcription factor 2α, 2β (TFAP2α and TFAP2β), cMyc, and PPAR α suppress USP22 expression. The specific binding of TFAP2α and TFAP2β to the USP22 promoter was further validated by binding site-directed mutagenesis and chromosome immunoprecipitation (Chip) assays. Furthermore, overexpression of TFAP2α and TFAP2β significantly up-regulate USP22 expression and downstream molecules of USP22, concurrently enhance the proliferation, migration and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. In addition, TFAP2 was demonstrated to be associated with a poor prognosis of NSCLC patients. Moreover, TFAP2 protein expression correlated with USP22 protein expression in human NSCLC tissues. Taken together, the findings of this study indicate TFAP2 is an important transcription factor driving USP22 gene expression, and it promotes the progression of NSCLC partially through enhancing USP22 expression. Citation Format: Keqiang Zhang, Ting Sun, Wendong Li, Rajendra Pangeni, Dan Raz. Transcription Factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2435.

ALDH1 expression predicts progression of premalignant lesions to cancer in Type I endometrial carcinomas

In type 1 endometrial cancer, unopposed estrogen stimulation is thought to lead to endometrial hyperplasia which precedes malignant progression. Recent data from our group and others suggest that ALDH activity mediates stemness in endometrial cancer, but while aldehyde dehydrogenase 1 (ALDH1) has been suggested as a putative cancer stem cell marker in several cancer types, its clinical and prognostic value in endometrial cancer remains debated. The aim of this study was to investigate the clinical value of ALDH1 expression in endometrial hyperplasia and to determine its ability to predict progression to endometrial cancer. Interrogation of the TCGA database revealed upregulation of several isoforms in endometrial cancer, of which the ALDH1 isoforms collectively constituted the largest group. To translate its expression, a tissue microarray was previously constructed which contained a wide sampling of benign and malignant endometrial samples. The array contained a metachronous cohort of samples from individuals who either developed or did not develop endometrial cancer. Immunohistochemical staining was used to determine the intensity and frequency of ALDH1 expression. While benign proliferative and secretory endometrium showed very low levels of ALDH1, slightly higher expression was observed within the stratum basalis. In disease progression, cytoplasmic ALDH1 expression showed a step-wise increase between endometrial hyperplasia, atypical hyperplasia, and endometrial cancer. ALDH1 was also shown to be an early predictor of EC development, suggesting that it can serve as an independent prognostic indicator of patients with endometrial hyperplasia with or without atypia who would progress to cancer (p = 0.012).

Yap activation in irradiated parotid salivary glands is regulated by ROCK activity.

Radiotherapy plays a major role in the curative treatment of head and neck cancer, either as a single modality therapy, or in combination with surgery or chemotherapy, or both. Despite advances to limit radiation-induced side-effects, the major salivary glands are often affected. This frequently leads to hyposalivation which causes an increased risk for xerostomia, dental caries, mucositis, and malnutrition culminating in a significant impact on patients’ quality of life. Previous research demonstrated that loss of salivary function is associated with a decrease in polarity regulators and an increase in nuclear Yap localization in a putative stem and progenitor cell (SPC) population. Yap activation has been shown to be essential for regeneration in intestinal injury models; however, the highest levels of nuclear Yap are observed in irradiated salivary SPCs that do not regenerate the gland. Thus, elucidating the inputs that regulate nuclear Yap localization and determining the role that Yap plays within the entire tissue following radiation damage and during regeneration is critical. In this study, we demonstrate that radiation treatment increases nuclear Yap localization in acinar cells and Yap-regulated genes in parotid salivary tissues. Conversely, administration of insulin-like growth factor 1 (IGF1), known to restore salivary function in mouse models, reduces nuclear Yap localization and Yap transcriptional targets to levels similar to untreated tissues. Activation of Rho-associated protein kinase (ROCK) using calpeptin results in increased Yap-regulated genes in primary acinar cells while inhibition of ROCK activity (Y-27632) leads to decreased Yap transcriptional targets. These results suggest that Yap activity is dependent on ROCK activity and provides new mechanistic insights into the regulation of radiation-induced hyposalivation.

Phenotyping of Disease-Initiating CD34+/CD38─ Stem Cells in BCR-ABL1─ MPN Reveals Expression of Multiple Cytokine Receptors and Resistance-Related Antigens

The classical BCR-ABL1-negative myeloproliferative neoplasms (MPN) are characterized by over-production of myeloid cells, disease-related mutations in certain driver-genes (JAK2, CALR, MPL) and an increased risk to transform to secondary acute myeloid leukemia (sAML). Although considered stem cell-derived neoplasms, little is known about the phenotype and functional properties of disease-initiating neoplastic stem cells (NSC) in MPN and sAML. Recent data suggest that MPN NSC reside in a CD34+ fraction of the malignant clone. Therefore, these cells are considered most critical target populations to be examined for expression of molecular and immunological targets with the aim to develop improved or even curative NSC-eliminating therapies, such as antibody-based or CAR-T cell approaches. Using a panel of monoclonal antibodies (n=40) and multicolor flow cytometry, we established the immunological phenotype and target expression profiles of putative CD34+/CD38─ NSC and CD34+/CD38+ progenitor cells in patients with polycythemia vera (PV, n=18), essential thrombocythemia (ET, n=29), primary myelofibrosis (PMF, n=38) and post-MPN sAML (n=11). In almost all patients, the putative MPN stem cells expressed the stem cell invasion receptors Hermes (CD44) and ADGRE5 (CD97), C1qR1 (CD93), the migration/adhesion receptor MIC2 (CD99), and the stem cell antigen AC133 (CD133). Contrasting normal stem cells, MPN NCS and sAML stem cells failed to express Thy-1 (CD90). Among the cytokine receptors tested, MPN NSC invariably displayed the TGFßR-related antigen endoglin (CD105), TPOR (CD110), SCFR KIT (CD117), IL-3RA (CD123), CXCR4 (CD184) and IGF-1R (CD221). NSC expressed particularly high levels of KIT and low levels of TPOR and IGF-1R. The IL-2RA (CD25) was identified on NSC in most patients with PMF and sAML, and in a few with ET, but not in patients with PV. Similarly, the GM-CSFR (CD116) was found to be expressed on NSC in most patients with PMF, a few with ET and no with PV. MPN NSC did not exhibit substantial amounts of M-CSFR (CD115), IL-3RB (CD131), FLT3 (CD135), NGFR (CD271) VEGFR-2 KDR (CD309), EPOR, MET or OSMRB. The CD34+/CD38+ MPN progenitor cells displayed a similar profile of cytokine receptors. In addition, MPN and sAML progenitor cells expressed IL-1RAP and CLL-1 in most donors examined. We next examined the expression of various immunological targets and resistance-mediating immune checkpoint antigens on NSC and MPN progenitor cells. In all MPN patients and all sAML patients tested, NSC were found to express substantial amounts of Siglec-3 (CD33) and low levels of Campath-1 (CD52) and MDR-1 (CD243). In addition, MPN NSC and sAML stem cells invariably displayed the "don't eat" me checkpoint IAP (CD47) and the classical checkpoint PD-L1 (CD274). Exposure to interferon-gamma (200 U/ml, 24 hours) resulted in an upregulation of PD-L1 on NSC. In a subset of patients, MPN NSC expressed low levels of HB15 (CD83). In contrast, MPN NSC and sAML stem cells failed to express B7-1 (CD80), B7-2 (CD86), PD-L2 (CD273) and PD1 (CD279). MPN progenitor cells and sAML progenitors expressed an identical profile of cell surface targets and checkpoint antigens. Finally, we confirmed the disease-initiating capacity of MPN stem- and progenitor cells (CD34+ cells) using primary PMF cells in xenotransplantation experiments employing NSGS mice expressing human interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF). After 28 weeks post injection, engraftment of human CD45+ cells in the bone marrow of NSGS mice was found in 15/15 mice injected with bulk mononuclear cells (MNC) containing CD34+ cells and in 0/15 NSGS mice injected with MNC depleted of CD34+ cells. Together, MPN NSC reside in a CD34+ fraction of the malignant clone and display a unique phenotype, including cytokine receptors, immune checkpoint molecules and other target antigens. The phenotypic characterization of neoplastic stem cells should facilitate their enrichment and the development of NSC-eradicating treatment concepts in MPN. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

Abstract 3779: Multiomic single cell analysis of normal human bone marrow identifies a unique stem and progenitor population that expands in AML

Abstract Hematopoietic stem and progenitor cells (HSPCs) can generate a diversity of blood cells throughout the human lifespan. Although these cells have been evaluated using both sorted (Corces et al. Nat. Genet. 2016) and single cell assays (Pellin et al Nat. Commun. 2019), there remains uncertainty in the degree of heterogeneity within HSPC subpopulations and their associated differentiation trajectories. The phenotypic diversity within HSPCs needs to be better characterized in order to understand the pathogenesis of blood disorders including hematologic malignancies. To address this need, we characterized healthy bone marrow mononuclear cells (BMMCs) with whole transcriptome analysis (WTA) and surface marker evaluation. We hypothesized that by utilizing concurrent RNA and surface marker analysis (n=35), we can improve HSPC clustering and characterize specific phenotypic states along unique hematopoietic differentiation trajectories. Three healthy BMMC samples were stained with antibody conjugated oligonucleotides (BD® Abseq) and analyzed using BD Rhapsody™. We filtered 8,070 high quality cells for 2,508 HSPCs, myeloid cells, and lymphocyte precursors. The antibody-derived tags (ADTs) obtained from BD® Abseq comprised 33 of the most informative features (n=2000) and resulted in more stable clustering as determined by within sum of squares (WSS = 898 verses 934 for WTA alone for 20 clusters). Additionally, we designed a targeted HSPC panel (n=500 genes) with BD® Abseq which identified similar cell clusters compared to the WTA alone and WTA plus ADT data (rand index = 0.88). HSPC clustering identified putative hematopoietic stem cell (HSC), common myeloid progenitor (CMP), and megakaryocyte-erythroid progenitor (MEP) clusters that expressed canonical surface markers. Of interest, we identified candidate novel myeloid, T-cell, and B-cell primed precursors and new surface marker expression gradients that align with specific differentiation trajectories. The results of this analysis will be presented. The HSPC clusters were converted into a signature matrix using Cibersortx (Newman et al. Nat. Biotechnol. 2019), and bulk acute myeloid leukemia (AML) and healthy samples were deconvolved into respective healthy cell clusters. We subsequently performed multivariate Cox proportional hazard analysis, and observed that high levels of healthy cluster 8 (H8; HR 3.40, 95% confidence interval 1.24-9.34), the candidate lymphoid-primed multipotent progenitor (LMPP), and low levels of healthy cluster 6 (H6; HR 0.26, 95% confidence interval 0.12-0.58), a candidate erythrocyte precursor, at diagnosis were associated with worse overall survival. Deconvolution using sorted healthy sub-populations (Corces et al. Nat. Genet. 2016) only identified erythrocyte precursors as statistically relevant (HR 0.27, 95% confidence interval 0.12-0.61). Of note, H8 had a distinct gene expression profile compared to that identified for the sorted LMPP sub-population using differential gene expression analysis. In summary, we identified novel cell type clusters and surface marker associations using combined single cell WTA and surface marker analysis (BD® Abseq). We were able to correlate cell types with both canonical and novel surface markers, and deconvolution analysis provided preliminary insights into their clinical relevance in AML. Citation Format: Asiri Ediriwickrema, Sreejith Ramakrishnan, Margaret Nakamoto, Smita Ghanekar, Bogdan Luca, Aaron Newman, Andrew Gentles, Ravindra Majeti. Multiomic single cell analysis of normal human bone marrow identifies a unique stem and progenitor population that expands in AML [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3779.

Derivation of oocyte-like cells from putative embryonic stem cells and parthenogenetically activated into blastocysts in goat

Germ cells are responsible for the propagation of live animals from generation to generation, but to surprise, a steep increase in infertile problems among livestock poses great threat for economic development of human race. An alternative and robust approach is essential to combat these ailments. Here, we demonstrate that goat putative embryonic stem cells (ESCs) were successfully in vitro differentiated into primordial germ cells and oocyte-like cells using bone morphogenetic protein-4 (BMP-4) and trans-retinoic acid (RA). Oocyte-like cells having distinct zonapellucida recruited adjacent somatic cells in differentiating culture to form cumulus-oocyte complexes (COCs). The putative COCs were found to express the zonapellucida specific (ZP1 and ZP2) and oocyte-specific markers. Primordial germ cell-specific markers VASA, DAZL, STELLA, and PUM1 were detected at protein and mRNA level. In addition to that, the surface architecture of these putative COCs was thoroughly visualized by the scanning electron microscope. The putative COCs were further parthenogenetically activated to develop into healthy morula, blastocysts and hatched blastocyst stage like embryos. Our findings may contribute to the fundamental understanding of mammalian germ cell biology and may provide clinical insights regarding infertility ailments.

Isolation of cancer stem-like cells from hepatocellular carcinoma cell line HepG2 by methods of magnetic-activated cell sorting, spheroid culture, and anti-tumor drug-resistant selection: A primary evaluation

Introduction: Recently reported data have suggested that only a small subset of cancer cells possess the capability to initiate malignancies. These observations were based on investigation of cells within the primary tumors displaying a distinct surface marker pattern. CD133 marker is a putative hematopoietic and neuronal stem cell marker, which is also considered to be a tumorigenic marker in brain, prostate and liver. Recent studies have shown that a small population of CD133-positive cells, indeed, exists in human hepatocellular carcinoma (HCC) cell lines and primary HCC tissues. This study was aimed at isolating the cancer stem-like cells from hepatocellular carcinoma cell line HepG2 using three different methods: magnetic-activated cell sorting (MACS), spheroid culture (SC), and anti-tumor drug (ATD) resistant selection.
 Methods: HepG2 hepatocellular carcinoma cells were expanded to yield enough cells that could be used to isolate cancer stem-like cells by these three methods. For MACS, cancer stem-like cells were sorted using anti-CD133 monoclonal antibody. For the second method, cancer stem-like cells were enriched by selection of anti-tumor drug resistance property. Lastly, for the third method, three-dimensional (3D) culture was used to enrich for the cancer stem-like cells. The cells obtained by the three methods were expanded to obtain an adequate number of cells for confirmation of CD133 expression.
 Results: The expression of CD133+ cells in the three methods was found to be different. In the MACS method, the expanded CD133+ sorted cells cultured through 2 passages only contained 0.40 % CD133+ cells. In the 3D spheroid cell culture, of the population of cells there were 38.39 % that were CD133+ cells. Lastly, in the anti-tumor drug (doxorubicin at 150 nM) resistant selection, 66.22 % were CD133+ cells.
 Conclusion: This study shows that isolation of HepG2 derived CD133+ population by culture with doxorubicin (150 nM) yields the highest efficiency and purity of the 3 methods studied.

Phenotypic Characterization of Leukemia-Initiating Stem Cells in Chronic Myelomonocytic Leukemia (CMML)

Chronic myelomonocytic leukemia (CMML) is a stem cell-derived hematopoietic neoplasm characterized by dysplasia, uncontrolled expansion of monocytic (progenitor) cells in the bone marrow (BM) and in the peripheral blood (PB), and an increased risk of progression to secondary acute myeloid leukemia (sAML). Patients with advanced CMML and sAML are often highly resistant to therapy and their prognosis is dismal. It is thought that drug resistance in myeloid malignancies is a quality of leukemia stem cells (LSC), but little is known about CMML-initiating and propagating LSC. We investigated the phenotype and functional behavior of putative CMML-initiating cells in 15 patients with CMML (7 females, 8 males; median age 73 years; range 45-82 years) and 6 with sAML following CMML (1 female, 5 males; median age 67.5 years; range 66-76 years). BM and/or PB samples were examined by multicolor flow cytometry using antibodies against CD34, CD38 and various additional surface markers and target antigens. In a subset of patients, putative stem and progenitor cells (CD34+ cells, CD34+/CD38─ cells and CD34+/CD38+ cells) were FACS-sorted to high purity (>95%) and were employed in xenotransplantation experiments or in drug testing experiments. We found that CMML-initiating and propagating LSC reside within the CD34+/CD38─ fraction of the malignant clone. Whereas highly purified CD34+ cells engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG) 1Eav/MloySzJ (NSGS) mice with full-blown CMML (engraftment rate 44.8±26.0%), no CMML was produced by the bulk of CD34- monocytic cells (engraftment rate 0.8±0.5%; p=0.002). CMML engraftment was also detectable when transplanting unselected mononuclear cells (engraftment rate 19.7±10.9%). By contrast, no leukemic engraftment was produced by CD38+ CMML fractions (engraftment rate 0.1±0.1%; p=0.003), indicating that the NSGS-repopulating CMML LSC reside specifically in a CD34+/CD38- fraction of the clone. In sAML, both the CD34+/CD38- cell fraction (engraftment rate 92.2±6.2%) and the CD34+/CD38+ fraction (engraftment rate 80.5±7.2%) produced engraftment with AML blasts in NSGS mice. In a next step, we established the cell surface phenotype of CD34+/CD38- LSC in CMML and sAML. As assessed by multicolor flow cytometry, CD34+/CD38- CMML cells invariably expressed CD33/Siglec-3, CD117/KIT, CD123/IL3RA, CD133/AC133, CD135/FLT3, and IL-1RAP. In a subset of patients, CMML LSC also expressed CD52 (9/11 patients; 81%), CD114 (3/7 patients; 43%), CD184 (9/12 patients; 75%), CD221 (8/11 patients; 73%) and/or CLL-1 (7/13 patients; 54%). CMML LSC did not express CD25 or CD26. However, in patients with sAML, LSC also displayed CD25 (median fluorescence intensity, MFI: CMML: 0.9 vs. sAML: 23.0; p<0.001). Compared to hematopoietic stem cells in normal BM (NBM), CMML LSC displayed slightly increased levels of CD117/KIT (MFI CMML: 32.5 vs. MFI NBM: 15.0; p=0.019), CD135/FLT3 (MFI CMML: 1.9 vs. MFI NBM: 0.8; p=0.001), CD184/CXCR4 (MFI CMML: 1.6 vs. MFI NBM 0.9; p=0.027), and IL-1RAP (MFI CMML: 1.6 vs. MFI NBM: 0.8; p=0.004). No correlations between surface-marker expression on LSC and the type of CMML (CMML-0/1/2 or dysplastic vs. proliferative CMML) or the clinical course were found. To confirm the clinical relevance of expression of surface target antigens on CMML LSC, we applied the CD33-targeted drug gemtuzumab-ozogamicin (GO). As assessed by combined staining for LSC (CD34+/CD38-) and AnnexinV/DAPI, incubation of CMML LSC with GO (0.001-1 µg/ml) resulted in dose-dependent apoptosis in all donors tested, and the same result was obtained in the monoblastic cell lines THP-1 (GO at 1 µg/ml: 94.2±1.5% vs. control: 12.7±2.2%, p<0.05) and Mono-Mac-1 (GO at 1 µg/ml: 56.4±12.1% vs. control: 10.1±0.6% p<0.05). In conclusion, LSC in CMML and sAML reside within CD34+/CD38─ cell populations that express distinct profiles of surface markers and target antigens. During progression of CMML into sAML, LSC apparently acquire CD25. Characterization of CMML LSC and LSC in sAML should facilitate their enrichment and the development of LSC-eradicating therapies. Disclosures Hoermann: Novartis: Honoraria; Roche: Honoraria. Sperr:Celgene: Consultancy, Honoraria; Novartis: Honoraria. Sill:Astex: Other: Advisory board; Novartis: Other: Advisory board; AbbVie: Other: Advisory board; Astellas: Other: Advisory board. Geissler:Novartis: Honoraria; AOP: Honoraria; Roche: Honoraria; Amgen: Honoraria; AstraZeneca: Honoraria; Ratiopharm: Honoraria; Celgene: Honoraria; Abbvie: Honoraria; Pfizer: Honoraria. Deininger:Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Ascentage Pharma: Consultancy, Honoraria; Fusion Pharma: Consultancy; TRM: Consultancy; Sangoma: Consultancy; Adelphi: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy. Valent:Pfizer: Honoraria; Blueprint: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Deciphera: Honoraria, Research Funding.

CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition.

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibition in vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as “melanoma initiating cells,” resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

Form of calf diet and the rumen. I: Impact on growth and development

Preweaning diet is known to affect rumen tissue appearance at the gross level. The objectives of this experiment were to investigate effects of different preweaning diets on the growth and development of the rumen epithelium and on putative rumen epithelial stem and progenitor cell measurements at the gene and cell levels. Neonatal Holstein bull calves (n = 11) were individually housed and randomly assigned to 1 of 2 diets. The diets were milk replacer only (MRO; n = 5) or milk replacer with starter (MRS; n = 6). Diets were isoenergetic (3.87 ± 0.06 Mcal of metabolizable energy per day) and isonitrogenous (0.17 ± 0.003 kg/d of apparent digestible protein). Milk replacer was 22% crude protein, 21.5% fat (dry matter basis). The textured calf starter was 21.5% crude protein (dry matter basis). Water was available ad libitum and feed and water intake were recorded daily. Putative stem and progenitor cells were labeled by administering a thymidine analog (5-bromo-2'-deoxyuridine, BrdU; 5 mg/kg of body weight in sterile saline) for 5 consecutive days and allowed a 25-d washout period. Calves were killed at 43 ± 1 d after a 6 h exposure to a defined concentration of volatile fatty acids. We obtained rumen tissue from the ventral sac and used it for immunohistochemical analyses of BrdU (putative stem and progenitor cells) and Ki67 (cell proliferation), gene expression analysis, and morphological measurements via hematoxylin and eosin staining. Epithelial stem and progenitor cell gene markers of interest, analyzed by real-time quantitative PCR, were β1-integrin, keratin-14, notch-1, tumor protein p63, and leucine-rich repeat-containing G protein-coupled receptor 5. Body growth did not differ by diet, but empty reticulorumens were heavier in MRS calves (MRS: 0.67 ± 0.04 kg; MRO: 0.39 ± 0.04 kg). The percentage of label-retaining BrdU basale cells was higher in MRO calves than in MRS calves (2.0 ± 0.3% vs. 0.3 ± 0.2%, respectively). We observed a higher percentage of basale cells undergoing proliferation in MRS calves than in MRO calves (18.4 ± 2.6% vs. 10.8 ± 2.8%, respectively). Rumen epithelial gene expression was not affected by diet, but the submucosa was thicker in MRO calves and the epithelium and corneum/keratin layers were thicker in MRS calves. Presumptive stem and progenitor cells in the rumen epithelium were identifiable by their ability to retain labeled DNA in the long term, changed proliferative status in response to diet, and likely contributed to observed treatment differences in rumen tissue thickness.

Abstract 2973: Growth response of human liver cancer cell lines to treatment with various agents targeting different members of the HER family and CDKs

Abstract Liver cancer is one of the most lethal types of cancer in the world. The heterogeneous nature of liver cancer, together with primary and secondary resistance to existing drugs are some of the important contributing factors. In the past three decades, abnormal expression and activation of human epidermal growth factor receptor (HER) family members have been reported in a wide range of epithelial cancers and several monoclonal antibody (mAb)-based drugs and small molecule tyrosine kinase inhibitors (TKIs) targeting the HER family members have been approved for the treatment of patients with a wide range of cancers. However, none of the HER inhibitors have yet been approved for the treatment of patients with liver cancer. In some studies, the expression of other members of the HER family or other growth factor receptors has been associated with the resistance to therapy with the HER inhibitors. In this study, we investigated the relative expression of all HER family members, other growth factor receptors (e.g. IGF-IR, C-MET) and the putative cancer stem cell biomarker CD44 in a panel of human liver cancer cell lines (LCCLs). We also investigated the sensitivity of these LCCLs to treatment with various agents including different types of TKIs with specificity to one or more members of the HER family, C-MET or IGFI-IR, as well as inhibitors of CDK4/6 (palbocicilib) and CDK1/2/5/9 (dinacicilib) and compared our data to those obtained with the FDA approved agents sorafenib and regorafenib for the treatment of liver cancer. While the great majority of the LCCLs were positive for the HER family members, overexpression of the EGFR, HER-3, and HER-4 were only present in SNU475, PLC/PRF5 and PLC/PRF5 LCCLs with mean fluorescence intensity (MFI) values of 620, 130 and 84 respectively. In contrast, the expression levels of C-MET and IGF-IR were the highest in SNU449 and HEPG-2 with MFI values of 95 and 99 respectively. Four of the seven LCCLs had overexpression of CD44 with the MFI values ranging from 329 to 704. With the exception of PLC/PRF5 which was found to be more sensitive to treatment with the pan-HER TKI afatinib (IC50 = 86nM) than treatment with sorafenib (IC50= 370nM) and regorafenib (372 nM), most of the other LCCLs were insensitive to treatment with the other types of HER inhibitors. Moreover, out of all the targeted therapies, the CDK inhibitors Dinacicilib and Palbocicilib were the most effective treatment for inhibiting the growth in vitro of these LCCLs. However, using linear regression analysis, we did not find any significant association between the expression level of different growth factor receptors and the response to therapy with various agents. Taken together our results show the heterogeneous nature of human LCCLs and the need for further investigation on the therapeutic potential of afatinib in combination with other agents in liver cancer. Citation Format: Ozlem H. Ozyamaci, Alan M. Seddon, Satvider Mudan, Helmout Modjtahedi. Growth response of human liver cancer cell lines to treatment with various agents targeting different members of the HER family and CDKs [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 2973.

Hyper-activation of Aurora kinase a-polo-like kinase 1-FOXM1 axis promotes chronic myeloid leukemia resistance to tyrosine kinase inhibitors

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL1 fusion protein. Accordingly, TK inhibitors have drastically changed the disease prognosis. However, persistence of the transformed hematopoiesis even in patients who achieved a complete response to TK inhibitors and the disease relapse upon therapy discontinuation represent a major obstacle to CML cure.MethodsThiostrepton, Danusertib and Volasertib were used to investigate the effects of FOXM1, AKA and Plk1 inhibition in K562-S and K562-R cells. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Quantitative reverse transcription (RT)-PCR was used to assess BCR-ABL1, FOXM1, PLK1 and AURKA expression. Protein expression and activation was assessed by Western Blotting (WB). Clonogenic assay were performed to confirm K562-R resistance to Imatinib and to evaluate cells sensitivity to the different drugs.ResultsHere we proved that BCR-ABL1 TK-dependent hyper-activation of Aurora kinase A (AURKA)-Polo-like kinase 1 (PLK1)-FOXM1 axis is associated with the outcome of Imatinib (IM) resistance in an experimental model (K562 cell line) and bone marrow hematopoietic cells. Notably, such a biomolecular trait was detected in the putative leukemic stem cell (LSC) compartment characterized by a CD34+ phenotype. Constitutive phosphorylation of FOXM1 associated with BCR-ABL1 TK lets FOXM1 binding with β-catenin enables β-catenin nuclear import and recruitment to T cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription complex, hence supporting leukemic cell proliferation and survival. Lastly, the inhibition of single components of AURKA-PLK1-FOXM1 axis in response to specific drugs raises the expression of growth factor/DNA damage-inducible gene a (GADD45a), a strong inhibitor of AURKA and, as so, a critical component whose induction may mediate the eradication of leukemic clone.ConclusionsOur conclusion is that AURKA, PLK1 and FOXM1 inhibition may be considered as a promising therapeutic approach to cure CML.

Su1047 – The Characterization of the Function of Glutathione Stransferase Omega 2 (GSTO2) Indicated the Presence of Putative Quiescent Stem Cells in Human Esophagus

Su1047 – The Characterization of the Function of Glutathione Stransferase Omega 2 (GSTO2) Indicated the Presence of Putative Quiescent Stem Cells in Human Esophagus

844 – Identification of Putative Cancer Stem Cells in Gastric Precancerous Lesions

844 – Identification of Putative Cancer Stem Cells in Gastric Precancerous Lesions

Effect of chemotherapy on cancer stem cells and tumor-associated macrophages in a prospective study of preoperative chemotherapy in soft tissue sarcoma

BackgroundCancer stem cells (CSC) may respond to chemotherapy differently from other tumor cells.MethodsThis study examined the expression of the putative cancer stem cell markers ALDH1, CD44, and CD133; the angiogenesis marker CD31; and the macrophage marker CD68 in soft tissue sarcomas (STS) before and after 4 cycles of chemotherapy with doxorubicin and ifosfamide in 31 patients with high-grade soft tissue sarcoma in a prospective clinical trial.ResultsNone of the markers clearly identified CSCs in STS samples. Macrophages represented a prominent component in viable tumor areas in pre-treatment STS biopsies, ranging from < 5 to > 50%. Furthermore, macrophages expressed CD44 and ALDH1. Macrophage density correlated with baseline maximum standardized uptake value (SUVmax) on fluoro-deoxyglucose positron emission tomography (PET) imaging. Pre-chemotherapy CD68 staining correlated positively with the baseline SUVmax, and negatively with the percent of viable tumor cells in post-chemotherapy resection samples. In particular, cases with more CD68-positive cells at biopsy had fewer viable tumor cells at resection, suggesting a better response to chemotherapy.ConclusionsIn conclusion, ALDH1, CD44, and CD133 are not likely to be useful markers of CSCs in STS. However, our observation of infiltrating macrophages in STS specimens indicates that these immune cells may contribute significantly to STS biology and response to chemotherapy, and could provide a potential target of therapy. Future studies should investigate macrophage contribution to STS pathophysiology by cytokine signaling.

High expression of aldehyde dehydrogenase 1 and tissue necrosis factor alpha may relate to chronic infection of buffalo mammary gland

Aldehyde dehydrogenase 1 (ALDH1) and hepatocyte nuclear factor 4A (HNF4A) are the putative mammary stem cell markers. Tissue necrosis factor alpha (TNFA) is involved in inflammation-associated carcinogenesis and cell proliferation. In this study, the gene expression profile of ALDH1, HNF4A and TNFA of buffalo mammary tissue using real-time quantitative PCR (RT-qPCR). Analysis of RT-qPCR data revealed that the relative expression (log2 fold change) of ALDH1 and TNFA during mastitis (vs. lactation) was increased (P < .05) by 2.98 and 4.71, respectively. The relative expression (log2 fold change; −7.39) of stem cell marker, HNF4A was decreased (P < .05) during mastitis. Histological analysis of mammary tissue during mastitis showed thickening of stroma and occasionally hyperplasia, predominantly in prepubertal and non-lactating animals. Although, the level of expression of these genes may vary, depending upon the physiological stage of the animals, however expression of ALDH1 and TNFA was high during mastitis. A systematic study on large samples of buffalo mammary tissue with appropriate comparisons needs to be evaluated with these markers for prognosis of buffalo mammary health.

The FAK inhibitor BI 853520 inhibits spheroid formation and orthotopic tumor growth in malignant pleural mesothelioma

No tyrosine kinase inhibitors are approved for malignant pleural mesothelioma (MPM). Preclinical studies identified focal adhesion kinase (FAK) as a target in MPM. Accordingly, we assessed the novel, highly selective FAK inhibitor (BI 853520) in 2D and 3D cultures and in vivo. IC50 values were measured by adherent cell viability assay. Cell migration and 3D growth were quantified by video microscopy and spheroid formation, respectively. Phosphorylation of FAK, Akt, S6, and Erk was measured by immunoblot. The mRNA expression of the putative tumor stem cell markers SOX2, Nanog, CD44, ALDH1, c-myc, and Oct4 was analyzed by qPCR. Cell proliferation, apoptosis, and tumor tissue microvessel density (MVD) were investigated in orthotopic MPM xenografts. In all 12 MPM cell lines, IC50 exceeded 5 μM and loss of NF2 did not correlate with sensitivity. No synergism was found with cisplatin in adherent cells. BI 853520 decreased migration in 3 out of 4 cell lines. FAK phosphorylation was reduced upon treatment but activation of Erk, Akt, or S6 remained unaffected. Nevertheless, BI 853520 inhibited spheroid growth and significantly reduced tumor weight, cell proliferation, and MVD in vivo. BI 853520 has limited effect in adherent cultures but demonstrates potent activity in spheroids and in orthotopic tumors in vivo. Based on our findings, further studies are warranted to explore the clinical utility of BI 853520 in human MPM.Key messagesResponse to FAK inhibition in MPM is independent of NF2 expression or histotype.FAK inhibition strongly interfered with MPM spheroid formation.BI 853520 has been shown to exert anti-tumor effect in MPM.

The ontogeny of myometrial stem cells in OCT4-GFP transgenic mouse model

BackgroundMyometrium, the muscular wall of the uterus, is an active organ markedly remodeled during a woman’s reproductive life, especially during pregnancy. Different studies using the 5-bromo-2′-deoxyuridine and side population methods in murine and human myometrium have suggested the presence of somatic stem cells in this tissue because of its remarkable regenerative capacity. Recently, our group has developed a surface-marker (Stro1/CD44)-specific approach to isolate and characterize myometrial somatic stem cells (SSCs) from humans and rats.ObjectiveIn this study, we aimed to identify and localize the putative myometrial stem cell population in the murine uterus by using the specific surface markers, Nanog/CD44.MethodsUteri from OCT4-GFP transgenic mice at different early-life time points were analyzed via single and double immunohistochemistry to co-localize myometrial stem cell marker CD44 with other general stemmness markers, e.g., Nanog and Oct-4. Finally, we correlated the frequency of myometrial stem cells in vivo with the expression of sex steroid hormone receptors, estrogen receptor α (ERα), and progesterone receptors A and B (PR A&B).ResultsNanog+/CD44+ stem cells were present in murine myometrium. Both stem cell markers were shown to co-localize with Oct-4 expression. Time-course experiments demonstrated that their percentages were significantly lower at the pre-sexual age of 1 week than at the sexually mature ages of 3 to 24 weeks. Importantly, both ERα and PR A&B were abundantly expressed in the myometrium at ages 1, 3 and 4 weeks.ConclusionsWe demonstrated that murine CD44+ myometrial cells have features of somatic stem cells with the expression of typical undifferentiated markers. Furthermore, our results suggest that myometrial stem cells are sex steroid hormone dependent, likely via paracrine pathway, and increase in numbers with reproductive maturity and rise in serum estrogen and progesterone levels around 3 weeks of age in mice. The abundance and early onset expression of ER/PR emphasize the vulnerability of neonatal myometrium to environmental endocrine disruptors which can potentially lead to permanent reprograming and adult onset of myometrial disorders such as uterine fibroids.