Cancer Stem Cells In Tumors Research Articles

IL-8-induced O-GlcNAc modification via GLUT3 and GFAT regulates cancer stem cell-like properties in colon and lung cancer cells.

Interleukin-8 (IL-8) is a pro-inflammatory chemokine that is associated with induction of chemotaxis and degranulation of neutrophils. IL-8 is overexpressed in many tumors, including colon and lung cancer, and recent studies demonstrated essential roles for IL-8 in tumor progression within the tumor microenvironment. However, the molecular mechanism underlying the functions of IL-8 in tumor progression is unclear. In this study, we found that IL-8 is overexpressed in colon and lung cancer cells with cancer stem cell (CSC)-like characteristics and is required for CSC properties, including tumor-initiating abilities. These findings suggest that IL-8 plays an essential role in the development of CSCs. We also showed that IL-8 stimulation of colon and lung cancer cells-induced glucose uptake and expressions of glucose transporter 3 (GLUT3) and glucosamine fructose-6-phosphate aminotransferase (GFAT), a regulator of glucose flux to the hexosamine biosynthetic pathway, resulting in enhancement of protein O-GlcNAcylation. We demonstrated that these events are required for the generation and maintenance CSC-like characteristics of colon and lung cancer cells. Moreover, an O-GlcNAcylation inhibitor, OSMI1, reduced CSC number and tumor development in vivo. Together, these results reveal that IL-8-induced O-GlcNAcylation is required for generation and maintenance of CSCs of colon and lung cancer cells and suggests this regulatory pathway as a candidate therapeutic target of CSCs.

Cytotoxicity of Luffa cylindrica (L.) M.Roem. extract against circulating cancer stem cells in hepatocellular carcinoma

Ethnopharmacological relevanceLuffa cylindrica (L.) M.Roem. has been recognized as traditional medicine for the treatment of various diseases like inflammatory diseases, diarrhea and viral infections. The usual parts used include fruit, seeds and leaves. Aim of the studyTo evaluate the anticancer activity of the hot water extract of the whole plant of Luffa cylindrica using circulating tumor cells and cancer stem cells isolated from the peripheral blood of hepatocellular carcinoma patients in vitro. Materials and methodsSeventy five adult patients who reported as stage II and III Hepatocellular carcinoma were selected. Blood samples were withdrawn and the circulating tumor cells were isolated from the whole blood. Tumor cells and cancer stem cells were detected and isolated by flow cytometric techniques. The isolated cell types were cultured and propagated in the tissue culture facility, the extract was tested on the isolated cells. ResultsLuffa cylindrica hot water extract has shown cytotoxic activity against circulating tumor cells of hepatocellular carcinoma especially the cells sub-population CD133+/CD44+ with little effect among CD133+/CD44- sub-population. ConclusionHot water extract of Luffa cylindrica whole plant could decrease the ratio of cancer stem cells in blood of HCC patients and may be used to minimize recurrence and metastasis in hepatocellular carcinoma patients.

1933P - Autofluorescence: A new marker for identifying cancer stem cells (CSCs) in primary tumors

1933P - Autofluorescence: A new marker for identifying cancer stem cells (CSCs) in primary tumors

Silencing of FTS increases radiosensitivity by blocking radiation-induced Notch1 activation and spheroid formation in cervical cancer cells

Increasing evidence(s) suggests that cancer stem cells (CSC) in tumours contribute to radio-resistance and recurrence. Notch plays an important role in the maintenance of CSC in many cancers including cervical cancer. Previously, we have reported the role of Fused Toes Homolog (FTS) in conferring radioresistance in cervical cancer cells in vitro and human subjects. The present study investigated the regulatory role of FTS in Notch signaling and maintenance of CSC upon irradiation of cervical cancer cells. The expression of Notch1, 2, 3, cleaved Notch1 and its downstream target Hes1, and spheroid formation was increased by irradiation. Silencing of FTS prevented the radiation-induced increase in the expression of Notch signaling molecules and spheroid formation. Immunoprecipitation showed FTS binds Notch1 and Hes1. Also in silico structural analysis identified putative residues responsible for the binding between FTS and Notch1. Spheroid formation and the expression of CSC markers, Nanog, Oct4A and Sox2 were greatly reduced by combining silencing of FTS and radiation. Taken together, these results suggest that FTS is involved in the regulation of irradiation-induced Notch signaling and CSC activation and can be used as a target to increase radiosensitivity in cervical cancer.

4Mu Decreases CD47 Expression on Hepatic Cancer Stem Cells and Primes a Potent Antitumor T Cell Response Induced by Interleukin-12.

The tumor microenvironment (TME) represents a complex interplay between different cellular components, including tumor cells and cancer stem cells (CSCs), with the associated stroma; such interaction promotes tumor immune escape and sustains tumor growth. Several experimental approaches for cancer therapy are focused on TME remodeling, resulting in increased antitumor effects. We previously demonstrated that the hyaluronan synthesis inhibitor 4-methylumbelliferone (4Mu) decreases liver fibrosis and induces antitumor activity in hepatocellular carcinoma (HCC). In this work, 4Mu, in combination with an adenovirus encoding interleukin-12 genes (AdIL-12), elicited a potent antitumor effect and significantly prolonged animal survival (p< 0.05) in an orthotopic HCC model established in fibrotic livers. In assessing the presence of CSCs, we found reduced mRNA levels of CD133+, CD90+, EpCAM+, CD44+, and CD13+ CSC markers within HCC tumors (p< 0.01). Additionally, 4Mu downregulated the expression of the CSC marker CD47+ on HCC cells, promoted phagocytosis by antigen-presenting cells, and, combined with Ad-IL12, elicited a potent cytotoxic-specific Tcell response. Finally, animal survival was increased when CD133low HCC cells, generated upon 4Mu treatment, were injected in a metastatic HCC model. In conclusion, the combined strategy ameliorates HCC aggressiveness by targeting CSCs and as a result of the induction of anticancer immunity.

Inhibition of HER2 Increases JAGGED1-dependent Breast Cancer Stem Cells: Role for Membrane JAGGED1.

Purpose: HER2-positive breast cancer is driven by cells possessing stem-like properties of self-renewal and differentiation, referred to as cancer stem cells (CSC). CSCs are implicated in radiotherapy, chemotherapy resistance, and tumor recurrence. NOTCH promotes breast CSC survival and self-renewal, and overexpression of NOTCH1 and the NOTCH ligand JAGGED1 predict poor outcome. Resistance to anti-HER2 therapy in HER2+ breast cancer requires NOTCH1, and that combination of trastuzumab and a gamma secretase inhibitor (GSI) prevents tumor relapse in xenograft models.Experimental Design: The current study investigates mechanisms by which HER2 tyrosine kinase activity regulates NOTCH-dependent CSC survival and tumor initiation.Results: Lapatinib-mediated HER2 inhibition shifts the population of HER2+ breast cancer cells from low membrane JAGGED1 expression to higher levels, independent of sensitivity to anti-HER2 treatment within the bulk cell population. This increase in membrane JAGGED1 is associated with higher NOTCH receptor expression, activation, and enrichment of CSCs in vitro and in vivo Importantly, lapatinib treatment results in growth arrest and cell death of JAGGED1 low-expressing cells while the JAGGED1 high-expressing cells continue to cycle. High membrane JAGGED1 protein expression predicts poor overall cumulative survival in women with HER2+ breast cancer.Conclusions: These results indicate that higher membrane JAGGED1 expression may be used to either predict response to anti-HER2 therapy or for detection of NOTCH-sensitive CSCs posttherapy. Sequential blockade of HER2 followed by JAGGED1 or NOTCH could be more effective than simultaneous blockade to prevent drug resistance and tumor progression. Clin Cancer Res; 24(18); 4566-78. ©2018 AACR.

High NRF2 level mediates cancer stem cell-like properties of aldehyde dehydrogenase\xa0(ALDH)-high ovarian cancer cells: inhibitory role of all-trans retinoic acid in ALDH/NRF2 signaling

Aldehyde dehydrogenase 1A1 (ALDH1A1) is one of cancer stem cell (CSC) markers, and high ALDH1 expression has been related to drug resistance and facilitated tumor growth. In this study, we investigated the potential involvement of nuclear factor erythroid 2-like 2 (NFE2L2/NRF2) in CSC-like properties of ALDH-high ovarian CSCs. Our experimental system, ALDH1A1-high (ALDH-H) subpopulation, was isolated and stabilized using doxorubicin-resistant ovarian cancer A2780 cells. ALDH-H exerted CSC-like properties such as drug resistance, colony/sphere formation, and enhanced tumor growth along with high levels of CSCs markers compared to ALDH1A1-low (ALDH-L). Levels of NRF2 and subsequent target genes substantially increased in ALDH-H cells, and the increase in ALDH1A1 and p62 was associated with NRF2 upregulation. ALDH1A1-silencing blocked increases in NRF2, drug efflux transporters, and p62, along with CSC markers in ALDH-H cells. The inhibition of p62, which was elevated in ALDH-H, suppressed NRF2 activation. High NRF2 level was confirmed in the ALDH1-high subpopulation from colon cancer HCT116 cells. The functional implication of NRF2 activation in ovarian CSCs was verified by two experimental approaches. First, CSC-like properties such as high CSC markers, chemoresistance, colony/sphere formation, and tumor growth were significantly inhibited by NRF2-silencing in ALDH-H cells. Second, all-trans retinoic acid (ATRA) suppressed ALDH1 expression, inhibiting NRF2 activation, which led to the attenuation of CSC-like properties in ALDH-H cells but not in ALDH-L cells. These results provide insight into the molecular basis of the ALDH1A1-mediated development of CSC-like properties such as stress/treatment resistance, and further suggest the therapeutic potential of ATRA in ALDH-high ovarian CSCs.

Breast cancer stem cells: Features, key drivers and treatment options.

The current view is that breast cancer is a stem cell disease characterized by the existence of cancer cells with stem-like features and tumor-initiating potential. These cells are made responsible for tumor dissemination and metastasis. Common therapies by chemotherapeutic drugs fail to eradicate these cells and rather increase the pool of cancer stem cells in tumors, an effect that may increase the likelyhood of recurrence. Fifteen years after the first evidence for a small stem-like subpopulation playing a major role in breast cancer initiation has been published a large body of knowledge has been accumulated regarding the signaling cascades and proteins involved in maintaining stemness in breast cancer. Differences in the stem cell pool size and in mechanisms regulating stemness in the different breast cancer subtypes have emerged. Overall, this knowledge offers new approaches to intervene with breast cancer stem cell activity. New options are particularly needed for the treatment of triple-negative breast cancer subtype, which is particularly rich in cancer stem cells and is also the subtype for which specific therapies are still not available.

Tumor suppressor LKB1 inhibits the progression of gallbladder carcinoma and predicts the prognosis of patients with this malignancy.

Gallbladder carcinoma(GBC) represents the most common fatal tumors of the biliary tract. The 3-year or 5-year survival rate for patients with this disease are 30 and 5%, respectively. Liver kinase B1 (LKB1), a primary upstream kinase of adenosine monophosphate-activated protein kinase (AMPK) necessary for maintaining cell metabolism and energy homeostasis, has been found to be an important tumor suppressor gene in recent years, and its inactivation has also found to be closely associated with tumor growth, metastasis and cancer stem cell (CSC) proliferation. Nevertheless, the function of LKB1 in GBC remains unclear. In this study, we found that the expression of LKB1 in GBC tissues was decreased compared with that in non-cancerous tissues. LKB1 overexpression suppressed the proliferation, metastasis and expansion of GBC CSCs. Mechanically, LKB1 suppressed GBC cell progression via the JAK/signal transducer and activator of transcription 3 (STAT3) pathway. The use of the JAK2 inhibitor, AZD‑1480, attenuated the suppressive effects of LKB1 overexpression on the growth, metastasis and self-renewal ability of the GBC cells, which further demonstrated that JAK/STAT3 was involved in the LKB1-induced suppression of GBC cell growth, metastasis and self-renewal ability. More importantly, the decreased expression of LKB1 was a predictor of a poor prognosis of patients with GBC. On the whole, our data indicate that LKB1 inhibits GBC cell growth, metastasis and self-renewal ability by disrupting JAK/STAT3 signaling, and may thus prove to be a novel prognostic biomarker for patients with GBC.

Abstract 165: Cancer stem cell phenotypes determine mechanisms of resistance to targeted therapies

Abstract Despite promising, and sometimes dramatic, efficacy of targeted therapies, primary and acquired resistance to targeted therapies limits their broad and long-term use in the clinic. Therefore, the ability to rationally design anticipatory or combination therapies as early in the treatment process as possible could significantly improve the utility of targeted therapies and cancer outcome. Here, we report that one can predict a priori the types of mutations that will occur in SHH pathway inhibitor treated medulloblastomas based on different cancer stem cell (CSC) phenotypes in each tumor. CSCs are a subset of cancer cells that are more resistant to cytotoxic therapies (chemo and radiation) than bulk tumor cells, and are responsible for tumor recurrence. Previously, we reported that cell-of-origin is a major driver of CSC phenotypes. In particular, we showed that CSCs retain epigenetic memory of their cells-of-origin and the mitogenic pathways that drive CSC proliferation and survival are reflective of their cells-of-origin. We also demonstrated that CSCs and bulk tumor cells do not necessarily depend on the same mitogenic/survival signaling pathways. An important implication of this finding is that targeted therapies selected based on bulk tumor analyses may not be effective in ablating CSCs in some tumors. Furthermore, the selective pressure imposed on CSCs in individual patient tumors by a targeted therapy will vary greatly, depending on whether the selected drug targets a critical pathway in CSCs and not just bulk tumor cells. Another clinically significant implication is that based on the resident CSC phenotype, we may able to predict whether individual SHH tumors will or will not acquire treatment-induced mutations in the SHH pathway upon SHH pathway inhibitors (SMOi) treatment. To test this hypothesis, we treated allografts of spontaneous Ptch;p53 mouse medulloblastomas arising from different cells-of-origins with SMOi's (LDE225 and GDC0449). While all tumors responded to SMOi's initially, most acquired resistance over time. We took an integrated genomics approach to identify molecular mechanisms of therapy resistance in these SMOi-resistant tumors. We report that acquired mutations in SHH pathway genes occurred only in tumors that contained CSCs that depended on the SHH pathway. In tumors where only the bulk tumor cells, but not CSCs, depended on SHH signaling, no acquired mutations in SHH pathway genes were detected. In sum, we report that CSCs drive mechanisms of therapy resistance and also provide the first proof-of-principle evidence that it is possible to predict the types of mutations that will occur in therapy-resistant tumors in advance of treatment, which can be exploited to design anticipatory therapies in the future. Citation Format: Joshy George, Yaohui Chen, Keiko Yamamoto, Scott Adamson, Jeff Chuang, Kyuson Yun. Cancer stem cell phenotypes determine mechanisms of resistance to targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 165.

Abstract 3460: p38-p14ARF-CtBP2 axis as a novel regulator of CSC phenotype and tumor cell dormancy

Abstract Introduction: Cancer cell dormancy and the cancer stem cell (CSC) phenotype are interlinked and play a crucial role in disease relapse. The pp38/pERK signaling ratio positively regulates cellular dormancy. We observed that heparin hexasaccharide (HS06) inhibited colon CSC self-renewal through isoform specific induction of pp38α/β, increasing the pp38/pERK ratio, and downregulation of C-terminal binding protein-2 (CtBP2) levels which is known to play a critical role in CSC growth. Hence, we hypothesized that HS06 inhibits CSCs self-renewal and prevents dormancy escape through regulation of CtBP2 in a manner dependent on p38 activation. Method: Spheroid culture of HT29 and HCT116 colon cancer cells was used for CSC self-renewal monitoring. Transient transfection with p38α, p14ARF, CtBP2 shRNA and/or expression vector with appropriate scrambled or vector controls was used to modulate cell signaling through this pathway. Standard western blotting, Q-PCR, flow cytometry, and immunoprecipitation were used to examine protein levels, their phosphorylation status and protein-protein interactions. Short term exposure of colon cancer cells (HT29) to chemotherapy (5-Fluorouracil (125μM) and oxaliplatin (5μM) (5-FUOX)) was used to study a transient dormancy-like state followed by growth escape. HT29 CSC (1x105 CD133+/CXCR4+ cells) induced xenografts were established s.c. in NCr nude mice were treated with HS06 (150mg/kg 3xwk., 3 weeks) and monitored for tumor growth, and xenografts were analyzed for CSC makers and p38-p14ARF-CtBP2 axis status. Results: HS06 inhibited CtBP2 levels but increased the expression of its negative regulator p14 ARF in vitro and in vivo in a manner dependent on p38 activation, as SB203580, a selective p38α/β inhibitor, as well as p38α shRNA, both near-completely reversed the effects of HS06 on p14ARF, CtBP2, and CSC self-renewal. This regulation of p14ARF and CtBP2 was post-transcriptional in nature. Moreover, reciprocal coimmunoprecipitation studies revealed a novel physical interaction between endogenous pp38 and p14ARF, which resulted in serine phosphorylation of p14ARF in a p38 activation-dependent manner that was critical for regulation of CtBP2 levels and CSC self-renewal. Using a chemotherapy induced cellular dormancy model, we observed that activation/upregulation of p38 and p14ARF prevented escape from dormancy in vitro, whereas increased CtBP2 had the opposite effect. Conclusion: We have discovered a novel p38-p14ARF-CtBP2 axis that plays a dual role in colon cancer CSC self-renewal and cellular dormancy. Agents targeting this pathway e.g., HS06 and CtBP2 inhibitors, represent novel therapeutic strategies to inhibit CSCs and prevent dormancy escape leading to reduced tumor relapse and/or cure. Citation Format: Rio S. Boothello, Nirmita J. Patel, Priyadarshan K. Damle, Chetna Sharon, Kranthi Kumar Chougoni, Umesh R. Desai, Steven R. Grossman, Bhaumik B. Patel. p38-p14ARF-CtBP2 axis as a novel regulator of CSC phenotype and tumor cell dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3460.

Abstract 4494: HMGA1 drives tumor progression and recruits cancer-associated fibroblasts in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors for which there are no effective therapies. Emerging evidence suggests that the tumor stroma interacts with the cancer cells to induce cancer stem cell properties and drive tumor progression. In PDAC, the fibroblast stroma also provides a dense barrier preventing cytotoxic therapy from reaching PDAC cells. We previously discovered that high levels of High Mobility Group A1 (HMGA1) protein predict decreased survival in primary PDAC. Here, we report a novel role for HMGA1-FGF19 in mediating tumor-stromal interactions and tumor progression. Silencing HMGA1 in PDAC cell lines or low-passge, patient-derived cells abruptly halts proliferation. Spindle-shaped, mesenchymal cells became reprogrammed into cuboidal, more epithelial-like cells. Sensitivity to gemcitabine was enhanced and colony formation, migration, invasion, and three-dimensional (3D) sphere formation were all disrupted in cells with HMGA1 knock-down. Silencing HMGA1 also disrupted xenograft tumorigenesis and depleted cancer stem cells/tumor-initiator cells in limiting dilution tumorigenicity assays. To elucidate underlying molecular mechanisms mediating these striking phenotypes, we performed RNA-seq after silencing HMGA1 in invasive, highly metastatic, low-passage patient-derived PDAC cells (10.7). Among the genes regulated by HMGA1 were those encoding proteins involved in tumor-stromal signaling, including the fibroblast growth factor 19 (FGF19). The FGF19 gene is highly expressed in GI tumors (liver, colon, PDAC) and transgenic mice overexpressing Fgr15 (the murine homolog) in hepatocytes develop hepatocellular carcinoma (HCC). FGF19 also correlates with poor outcomes in human HCC and colon cancer, although it's role in PDAC was unknown. Here, we found that FGF19 expression is dependent upon HMGA1 in 3 different PDAC cell lines; silencing HMGA1 represses FGF19 in these cells. HMGA1 also binds directly to the FGF19 promoter at 2 predicted DNA binding sites as assessed by chromatin immunoprecipiation. To determine whether FGF19 plays a functional role in HMGA1-mediated tumor progression and cancer stem cell properties, we silenced FGF19 in PDAC cells. Similar to our results with HMGA1, silencing FGF19 impaired PDAC growth and 3D sphere formation in vitro. Because fibroblast growth factors interact with fibroblasts, we determined whether the HMGA1-FGF19 pathway was involved in tumor cell – stromal crosstalk. PDAC 10.7 cells recruit cancer-associated fibroblasts (CAFs) in a co-culture system, although this recruitment was abrogated when HMGA1 was silenced. CAF migration was also disrupted by anti-human FGF19 neutralizing antibodies. Together, these findings indicate that HMGA1 drive tumor progression and cancer stem cell properties through FGF19 and suggest that targeting the HMGA1-FGF19 pathway maybe efficaceous in PDAC. Citation Format: Shuai Shuai, Lingling Xian, Tait Huso, Karen Reddy, Linda Resar. HMGA1 drives tumor progression and recruits cancer-associated fibroblasts in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4494.

PO-282 SMAR1 transcriptionally repress hTERT and alters the stem cell population in colorectal cancer cells

IntroductionOne of the distinguishing features that set the cancer cell apart is its ability for indefinite proliferation. Cancer progression is accompanied by perturbations in the chromatin structure that results in inappropriate gene expression and genomic instability. Aberrant activation of signalling pathways in cancer cells lead to a cascade of events resulting in ‘replicative immortality’. One of the mechanisms by which cancer cells keep dividing is by the reactivation of enzyme telomerase (hTERT). hTERT reactivation confers tumour cell with various advantages like protection against DNA damaging agents, maintenance of stem cell pool, increased angiogenesis, etc. This cancer stem cell (CSC) population then remains quiescent owing to its radio and chemo-resistant nature and leads to cancer relapse.Material and methodsWestern Blotting, Real time PCR, Co-Immunoprecipitation, Chromatin Immunoprecipitation, Telomerase Repeat Amplification Protocol (TRAP) Assay, Polyps generation in Balb/c mice.Results and discussionsOur study elucidates the role of a Martix Attachment Region Binding Protein (MARBP), SMAR1 as a transcriptional repressor of hTERT. SMAR1 directly interacts with HDAC1/mSin3a co-repressor complex and bind to the hTERT promoter. This binding of SMAR1 brings about Wnt/β-Catenin mediated repression of hTERT. Interestingly we find that occupancy of HDAC1 on hTERT promoter is SMAR1-dependent. The recruitment of repressor complex on the hTERT promoter results in the deacetylation of histones and an increase in the H3K9me2 mark.This helps maintain a repressive environment on the hTERT promoter and alters the occupancy of PolII on the promoter. We also find that knock-down of SMAR1 promotes CD133+ CSC’s and SMAR1 over-expression inhibits CD133+ CSC’s. Moreover, CD133high and CD133low populations exhibit differential SMAR1 and hTERT expression. We generated polyps in Balb/c mice using azoxymethane (AOM)/dextran sodium sulphate (DSS). When the levels of SMAR1 and hTERT were assessed; polyps expressed lower levels of SMAR1 and elevated hTERT as compared to the adjacent non-cancerous tissue.ConclusionWe show that SMAR1 transcriptionally repress hTERT. Given that hTERT reactivation is central for tumour development, progression and cancer stem cell maintenance, this regulation by SMAR1 represents an important mechanism of tumour suppression and inhibition of cancer stem cell trait.

Abstract 5607: Tissue factor is a novel oncotarget for immunotherapy of triple-negative breast cancer using a second generation ICON in orthotopic mouse models of human and murine TNBC cell line- and patient-derived xenografts

Abstract Triple-negative breast cancer (TNBC) is often associated with BRCA1 and BRCA2 mutation and is a leading cause of breast cancer death. Due to the lack of validated target molecules, there is currently no approved targeted therapy for TNBC. Tissue factor (TF) is a common yet specific surface target receptor for cancer cells, tumor vascular endothelial cells and cancer stem cells in several types of solid cancers. Here we report evidence supporting the idea that TF is a novel surface target for TNBC patients and cancer lines with BRCA1 and BRCA2 mutations. We showed that TF is over-expressed on TNBC cells and tumor neovasculature in greater than 85% of TNBC patients (n=14) when using standard paraffin-embedded tumor tissues and in nearly 60% of TNBC patients (n=157) when employing tissue microarray slides, but was not detected in adjacent normal breast tissue. We also describe the development of a second-generation TF-targeting immunoconjugate with improved efficacy and higher safety threshold compared to the original immunoconjugate. We showed that L-ICON1 is effective in killing TNBC cells via antibody-dependent cell-mediated cytotoxicity in vitro and in treating human and murine TNBC cell line- and patient-derived xenografts in vivo in preclinical orthotopic mouse models. These results suggest that TF is a novel oncotarget in TNBC and targeting TF may constitute a novel targeted immunotherapy for TNBC patients. The findings in this preclinical study warrant further clinical investigation. Citation Format: Zhiwei Hu, Rulong Shen, Amanda Campbell, Elizabeth McMichael, Lianbo Yu, Bhuvaneswari Ramaswamy, Cheryl A. London, Tian Xu, William E. Carson. Tissue factor is a novel oncotarget for immunotherapy of triple-negative breast cancer using a second generation ICON in orthotopic mouse models of human and murine TNBC cell line- and patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5607.

PO-280 Hypoxia-induced PLD2 promotes tumorigenesis and stem cell-like properties in tumour cells

IntroductionPhospholipase D (PLD) hydrolyzes phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes, and it is overexpressed in a wide variety of cancers. In solid tumours, PLD2 has been associated to hypoxic regions and it has been purposed to mediate postranslational regulation of HIF-α, but its contribution to protein stabilisation or degradation is not clear. Moreover, hypoxia has a key role in tumour population heterogeneity and cancer stem cells increase and it has also been associated with increased malignancy, poor prognosis and resistance to radiotherapy and chemotherapy.Material and methodsTo characterise the implication of PLD2 in tumorigenesis and the increase of stem cell-like properties under hypoxia conditions, we generated two PLD2 knock-down colon cancer cell lines from HCT116 and LS180 commercial cell lines. We also generated PLD2 lipase mutant cell lines. We checked PLD2 protein and mRNA levels under normoxia and hypoxia conditions, as well as tumorigenesis, using different approaches in vitro: growth curves, colony formation assays, percentage of holoclones and capacity to generate tumorspheres. In addition, we analysed several stem cell markers by FACS analysis and RT-qPCR.Results and discussionsWe first show that the increase of PLD2 under hypoxia conditions depends on its lipase activity. Hypoxia-induced PLD2 increases cell growth and anchorage-independent cell growth. Additionally, we show that hypoxia-induced PLD2 increases the stem cell-like properties of cancer cells, including clonogenicity and the capacity to form tumorspheres.ConclusionAltogether, our results demonstrate that hypoxia-induced PLD2 increases tumorigenesis and stem cell-like properties in colon cancer cells.

Abstract 4660: Tumor multicomponent targeting nanoparticle library for personalized cancer therapy &amp; imaging

Abstract The fundamental limitations of conventional cancer treatment have promoted the development and application of nanoengineering platforms for achieving effective patient outcomes. Over a time, considerable clinical success has been accomplished in nanotechnology field but there are many challenges limiting the progress of nanomedicine from bench to the beside. The highly heterogeneous tumor environment, an inadequate understanding of interactions between tumor biomarkers and nanomedicine, and reproducibility in synthesis required for clinical translation are some of the major challenges. Thus, there is an urgent need to redirect our focus of nano-drug carrier that can target multiple components of tumor including, tumor epithelial cells, tumor angiogenic vessels, tumor hypoxia and stromal cells. To achieve this, we developed a library of various tumor biomarker targeting ligands, and drug and imaging agents encapsulated in nanoparticles (NPs). Finally, the library of targeting ligands was conjugated with NPs using reagent free ‘click' chemistry. This strategy will serve as an “off-the-shelf ready to configure drug formulation" that can be engineered based on a mix-and-match strategy for individual patient needs. It is envisaged that the patient can walk in the clinic and be given the targeted drug that is personalized to their need. For the proof-of-concept, we evaluated tumor selectivity of multiple cancer biomarker targeting oligo-micelles (OMs), iron oxide and lipid NPs in various patient derived tumor xenograft (PDx) models, such as non-small cell lung and triple negative breast cancer. We observed that the dual tumor hypoxia and cancer stem cell (CSC) targeting rod shaped OM has 8-fold higher tumor core penetration compared to non-targeted OM in these PDx tumors, as obtained from NIR-fluorescence imaging. This OM has significantly higher tumor vs. liver ratio and they follow tumor specific receptor competition. The tumor growth inhibition of drug loaded OM is superior compared to free drug. This approach demonstrated significant improvement of tumor stroma penetration with low healthy organ uptake of NPs portending more promising potentials for selective cancer treatment. Citation Format: Samaresh Sau, Hashem Alsaab, Katyayani Tatiparti, Ketki Bhise, Arun Rishi, Arun K. Iyer. Tumor multicomponent targeting nanoparticle library for personalized cancer therapy &amp; imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4660.

LW106, a novel indoleamine 2,3-dioxygenase 1 inhibitor, suppresses tumour progression by limiting stroma-immune crosstalk and cancer stem cell enrichment in tumour micro-environment.

Indoleamine 2,3-dioxygenase 1 (IDO1) is emerging as an important new therapeutic target for treatment of malignant tumours characterized by dysregulated tryptophan metabolism. However, the antitumour efficacy of existing small-molecule inhibitors of IDO1 is still unsatisfactory and the underlying mechanism remains largely undefined. Hence, we discovered a novel potent small-molecule inhibitor of IDO1, LW106, and studied its antitumour effects and the underlying mechanisms in two tumour models. C57BL6 mice, athymic nude mice or Ido1-/- mice were inoculated with IDO1-expressing and -nonexpressing tumour cells and treated with vehicle, epacadostat or increasing doses of LW106. Xenografted tumours, plasma, spleens and other vital organs were harvested and subjected to kynurenine/tryptophan measurement and flow cytometric, histological and immunohistochemical analyses. LW106 dose-dependently inhibited the outgrowth of xenografted tumours that were inoculated in C57BL6 mice but not nude mice or Ido1-/- mice, showing a stronger antitumour efficacy than epacadostat, an existing IDO1 inhibitor. LW106 substantially elevated intratumoural infiltration of proliferative Teff cells, while reducing recruitment of proliferative Treg cells and non-haematopoietic stromal cells such as endothelial cells and cancer-associated fibroblasts. LW106 treatment resulted in a reduced subpopulation of cancer stem cells (CSCs) in xenografted tumours in which fewer proliferative/invasive tumour cells and more apoptotic tumour cells were observed. LW106 inhibits tumour outgrowth by limiting stroma-immune crosstalk and CSC enrichment in the tumour micro-environment. LW106 has potential as a immunotherapeutic agent for use in combination with immune checkpoint inhibitors and (or) chemotherapeutic drugs for cancer treatment.

Cancer stem cells in colorectal tumors of various extents.

e15659Background: The purpose of the study was to assess an association between the tumor invasion and levels of cancer stem cells (CD44+ and CD133+) in colorectal cancer. Methods: The study includ...

Interplay between inflammatory tumor microenvironment and cancer stem cells.

Cancer stem cells (CSCs), which have a close connection with tumor microenvironment, play a pivotal role in tumorigenesis, tumor progression, and metastasis. The inflammatory microenvironment is an essential component of tumor microenvironment. In the recent years, many studies have demonstrated that the inflammatory microenvironment induces the initiation of tumors, and contributes to the process of the progression of tumors, as well as metastasis. In this review, we summarize the relationship between CSCs and inflammatory components, such as inflammatory cytokines (IFNs, TNF, IL-6, IL-17) and inflammatory cells (myeloid-derived suppressor cells, tumor-associated macrophages). To illuminate the key factors that exert important actions in the tumor process would be important to improve the clinical outcome of the treatment for different types of cancer.

Involvement of partial EMT in cancer progression.

The epithelial-mesenchymal transition (EMT) provides an outstanding example of cellular plasticity during embryonic development and cancer progression. During EMT in embryonic development, epithelial cells lose all vestiges of their epithelial origin and acquire a fully mesenchymal phenotype, known as complete EMT, which is typically characterized by a so-called cadherin switch. Conversely, during EMT in cancer progression, cancer cells that originate from epithelial cells exhibit both mesenchymal and epithelial characteristics, that is the hybrid E/M phenotype in a process known as partial EMT. Partial EMT in cancer cells is thought to enhance their invasive properties, generate circulating tumour cells and cancer stem cells, and promote resistance to anti-cancer drugs. These phenotypic changes are regulated by extracellular matrix components, exosomes and soluble factors, which regulate several transcription factors known as EMT transcription factors. In this review, I summarize our current understanding of the EMT program during cancer progression.