Mesenchymal Subpopulations Research Articles

TNF-α-Induced Bone Marrow Stromal Progenitor Alterations Enhance Leukemic Stem Cell Growth and Treatment Resistance Via Increased CXCL1-CXCR2 Signaling

Mesenchymal cells within the bone marrow (BM) microenvironment play an important role in regulation of hematopoietic stem cell (HSC) fate. However, the effects of leukemia development on distribution and function of mesenchymal cell subpopulations are not well understood. Here we used the SCL-tTA-BCR-ABL transgenic CML mouse model to examine how CML development affected murine BM mesenchymal subpopulations within a recently delineated skeletal stem cell (SSC) hierarchy, and evaluate how mesenchymal subpopulations affected HSC and leukemia stem cell (LSC) growth.We observed a significant increase in bone-forming “Thy” and stroma-forming “6C3” progenitor subsets in CML compared to normal BM. CML Thy and 6C3 progenitors demonstrated increased proliferation and CFU-F potential. In addition, CML Thy cells exhibiting increased osteogenic potential whereas CML 6C3 cells showed increased adipogenic potential compared to their normal counterparts.CML LSC and normal HSC were cocultured with Thy and 6C3 cells, purified from CML and normal mice for 3 days and transplanted into irradiated normal recipients to evaluate long-term engraftment. Normal HSC engraftment was enhanced by coculture with normal Thy and 6C3 cells but not by their CML counterparts. On the other hand, LSC engraftment was enhanced by CML 6C3 cells compared to normal 6C3 cells, but not by CML Thy cells compared to normal Thy cells. These results indicate that CML stromal progenitors demonstrate enhanced support of LSC and reduced support of normal HSC.Q-PCR analysis showed that expression of major hematopoietic regulatory molecules, including CXCL12, G-CSF, SCF, IL-1, IL-6, and IGF-1, was significantly reduced in CML 6C3 and Thy progenitors.RNA-Seq analysis demonstrated that expression of TNFaand NF-kbrelated gene sets was significantly increased in CML compared with normal 6C3 cells. Ligand-receptor interactome analysis, based on differential gene expression in LSC and normal HSC, and CML and normal 6C3 cells, revealed upregulation of the chemokines CXCL1 and CXCL5 in CML 6C3 cells, and of their cognate receptor CXCR2 in LSC. We have previously shown that TNFalevels are increased in CML compared to normal BM (Zhang et al., 2012). Here we found that treatment of WT mice with TNFα led to expansion of 6C3 cells and increased CXCL1 expression on 6C3 cells. In contrast, treatment of CML mice with anti-TNFα antibodies led to reduction in 6C3 cell numbers and reduced CXCL1 expression in 6C3 cells. These results support a critical role for TNFα signaling in expansion and increased CXCL1 expression by stromal progenitors in CML BM.We evaluated the role of paracrine CXCL1-CXCR2 signaling in growth and TKI resistance of LSC. Treatment with CXCL1 and CXCL5 resulted in expansion of LSC and leukemic progenitors respectively, and this effect was blocked by the CXCR2 inhibitor SB225002. Treatment with SB225002 significantly reduced proliferation of LSC cocultured with CML 6C3 cells. SB225002 administration to mice engrafted with CML cells resulted in significant reduction in peripheral blood WBC counts, neutrophil percentage, and leukemic short-term HSC (STHSC) and granulocyte-macrophage progenitors (GMP) in the BM. The combination of SB225002 and the TKI Nilotinib (50mg/kg) resulted in significantly greater reduction in peripheral blood WBC and neutrophils, and in BM LSC compared to Nilotinib or SB225002 alone.SB225002 treatment also significantly reduced proliferation and enhanced apoptosis of human CML CD34+CD38- cells cocultured with human CML BM mesenchymal stromal cells, and that the combination of SB225002 and Nilotinib significantly enhanced apoptosis and inhibited proliferation of CML CD34+CD38- cells compared to Nilotinib alone.We conclude that increased TNFα signaling results in expansion of stromal progenitors in CML BM, which differentially support LSC compared to normal HSC. TNFα-signaling leads to overexpression of CXCL1 by stromal progenitors, which interacts with CXCR2 overexpressed on LSC to enhance their growth. Inhibition of CXCR2 signaling reduces LSC proliferation and survival, and enhances LSC elimination in combination with TKI. These observations support further exploration of targeting of CXCL1-CXCR2 interactions as a novel and effective strategy to target BM microenvironment-protected TKI-resistant LSC. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4968: Tumor innate immunity primed by specific interferon stimulated endogenous retroviruses

Abstract Introduction: Tumor cell heterogeneity is a key determinant of cancer progression and drug resistance, which is often mediated by mesenchymal cell subpopulations. While these subclones can secrete growth factors, chemokines and cytokines, the immune signaling networks that fuel this pro-tumorigenic state remain incompletely defined. Elucidating what underlies this state would provide insights into tumor biology and inform clinical strategies to improve anti-cancer therapies. Methods: Because of their well-defined nature, we used the phenotypically distinct H69M and H69AR Small Cell Lung Cancer (SCLC) mesenchymal subclones to uncover a novel mechanism of dysregulated innate immune signaling as compared with parental neuroendocrine H69 cells. Analysis of gene signatures across TCGA and CCLE databases, functional studies in additional cell lines, and ex vivo testing of patient-derived organotypic tumor spheroids (PDOTS) were conducted to determine the broader relevance across human cancers. Results: We discovered a novel epigenetically regulated subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in mesenchymal cancer subpopulations. Stimulated 3 Prime Antisense Retroviral Coding Sequences (SPARCS) are oriented inversely in 3'UTRs of certain interferon-inducible genes and silenced by EZH2. De-repression of these loci resulted in dsRNA generation following IFNγ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' LTR of the antisense ERV. We found that dsRNA sensing preferentially by MAVS fuels activation of TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction across specific human tumors and cell lines is tightly associated with downregulation of chromatin modifying enzymes, including EZH2, a mesenchymal AXL positive cell state, and B2M and MHC class 1 antigen expression. SPARCS high tumors were marked by immune infiltration, but also exhibited multiple features of tumor immune suppression. IFNγ treatment of PDOTS with de-repressed SPARCS markedly enhanced CXCL10 production and sensitized them to PD-1 blockade. Conclusions: Together, these data unveil a novel subclass of ERVs whose de-repression triggers pathologic innate immune signaling in cancer, with potentially important implications for cancer immunotherapy. Citation Format: David A. Barbie. Tumor innate immunity primed by specific interferon stimulated endogenous retroviruses [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 4968.

An integrated flow cytometry-based platform for isolation and molecular characterization of circulating tumor single cells and clusters

Comprehensive molecular analysis of rare circulating tumor cells (CTCs) and cell clusters is often hampered by low throughput and purity, as well as cell loss. To address this, we developed a fully integrated platform for flow cytometry-based isolation of CTCs and clusters from blood that can be combined with whole transcriptome analysis or targeted RNA transcript quantification. Downstream molecular signature can be linked to cell phenotype through index sorting. This newly developed platform utilizes in-line magnetic particle-based leukocyte depletion, and acoustic cell focusing and washing to achieve >98% reduction of blood cells and non-cellular debris, along with >1.5 log-fold enrichment of spiked tumor cells. We could also detect 1 spiked-in tumor cell in 1 million WBCs in 4/7 replicates. Importantly, the use of a large 200μm nozzle and low sheath pressure (3.5 psi) minimized shear forces, thereby maintaining cell viability and integrity while allowing for simultaneous recovery of single cells and clusters from blood. As proof of principle, we isolated and transcriptionally characterized 63 single CTCs from a genetically engineered pancreatic cancer mouse model (n = 12 mice) and, using index sorting, were able to identify distinct epithelial and mesenchymal sub-populations based on linked single cell protein and gene expression.

Molecular Profiling of Pooled Circulating Tumor Cells from Prostate Cancer Patients Using a Dual-Antibody-Functionalized Microfluidic Device.

To capture both epithelial and mesenchymal subpopulations of CTCs at different metastatic stages of PCa patients, here we constructed a novel dual-antibody-functionalized microfluidic device by employing antibodies against PSMA and EpCAM. In vitro experiments with the dual capture system for capturing both LnCAP and LnCAP-EMT cells have shown significantly enhanced capture efficiency as compared to that of the EpCAM single capture system. Furthermore, the dual capture system could successfully identify CTCs in 20 out of 24 (83.3%) PCa patients, and the CTCs counts from the dual capture system were statistically correlated with the TNM stage of patients ( P < 0.05), while conventional diagnostic methods, such as serum PSA level and Gleason score, failed to correlate to patient TNM stages. To further explore potential clinical application of our dual capture system, captured CTCs were recovered and subjected to qRT-PCR to quantify known factors involved in PCa development and therapy. The results demonstrated that the combined detection of SChLAP1 and PSA in CTCs is a potential marker for identifying patients with metastatic PCa, while detection of AR and PD-L1 in CTCs may have the potential to determine the sensitivity of PCa patients to androgen deprivation therapy and immunotherapy, respectively. Taken together, the dual-antibody-functionalized microfluidic device established in our study overcomes the limitations of some CTC capture platforms that only detect epithelial or mesenchymal CTCs in PCa patients, and detection of the PCa-related RNA signatures from purified CTCs holds great promise to offer warnings for early metastasis of PCa and may provide guidance for therapy decisions.

Transcriptionally and Functionally Distinct Mesenchymal Subpopulations Are Generated from Human Pluripotent Stem Cells.

SummaryVarious mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFRβ), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies.

Abstract B123: A mesenchymal subset of cancers with elevated ZEB1 expression is sensitive to low doses of silver nanoparticles

Abstract The epithelial-to-mesenchymal transition (EMT) is an evolutionarily conserved process that is required for normal development. However, EMT is often dysregulated and hijacked by cancers during tumor progression. Cancer cells that have undergone EMT and acquired a mesenchymal phenotype harbor several distinct traits, including increased tumor initiating properties, augmented motility, increased metastatic properties, and a higher incidence of resistance to therapy. Therefore, treatment regimens specific to mesenchymal cancer cells would be advantageous. In this study, we discovered that a mesenchymal subpopulation of breast, ovarian, lung, colorectal, and prostate cancer cell lines with high expression of the transcription factor ZEB1 (zinc finger E-box binding homeobox 1) and low expression of epithelial genes repressed by ZEB1 including ESRP1 (epithelial splicing regulatory protein 1) and CDH1 (E-cadherin) are an order of magnitude (&amp;gt;10-fold) more sensitive to exposure to silver nanoparticles (AgNPs) than cells expressing only low levels of ZEB1. This finding is significant because: (i) This is the first time a biomarker has been identified that is predictive of a positive response to any form of engineered nanomaterial; (ii) ZEB1 expression is a marker of poor clinical prognosis, and therefore identification of improved therapeutics for this poor outcome patient population is desirable; (iii) this work establishes that there is a shared and exploitable vulnerability across multiple cancer types that is independent of tissue origin and identifiable based upon mRNA or protein quantification of ZEB1. We establish that the ZEB1high selective cytotoxic properties of biocompatible AgNPs are not achieved using ionic silver, and therefore is one of the first examples of a “new to nano” cytotoxic property. Mechanistically, we find that AgNPs are rapidly ionized in ZEB1high cells, leading to endoplasmic reticulum stress, DNA damage, and apoptotic cell death. Furthermore, we show that AgNPs do not disrupt the normal architecture of breast acini in 3D cell culture, nor do they cause DNA damage. In contrast, the same doses of AgNPs cause extensive DNA damage and apoptosis in ZEB1high breast tumor nodules produced in 3D culture. Induction of EMT using TGF-β increases sensitivity to AgNPs and, conversely, knockdown of ZEB1 desensitizes cells to AgNP therapy. Finally, we demonstrate that systemic administration of AgNPs is safe and effective for treatment of ZEB1high breast cancer orthotopic xenografts in mice. In conclusion, our work provides evidence that a therapeutic window exists for the safe use of AgNPs as a precision medicine for patients with ZEB1-expressing tumors regardless of tissue origin. Citation Format: Cale D. Fahrenholtz, Jessica Swanner, James Sears, Katherine Cook, Pierre-Alexandre Vidi, Ravi Singh. A mesenchymal subset of cancers with elevated ZEB1 expression is sensitive to low doses of silver nanoparticles [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B123.

Abstract 1334: Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in triple negative breast cancer

Abstract Recent studies correlate Hedgehog (Hh) signaling with reduced survival rates in triple negative breast cancer (TNBC) patients. Activation of hedgehog signaling in the adjacent mesenchyme has been shown to promote tumor growth and it is a poor prognosis factor for TNBC. We developed a novel tumor-mesenchymal in vitro model of hedgehog signaling in TNBC to evaluate the role of mesenchymal cell sub-populations in the proliferative potential and stem cell markers of breast cancer cells using a custom designed multiwell array. As a source of mesenchymal cell sub-populations we evaluated myofibroblasts, mesenchymal stem cells derived from bone marrow or adipose tissue and tumor cells that undergo epithelial-mesenchymal transition (EMT). Tumor cells were culture with 1 or 2 mesenchymal cell sub-populations in adjacent compartments +/- SHH ligand for 72-96hrs. Active Hh signaling was confirmed by up-regulation of main Hh target genes (e.g. Gli1, Patch1). Paracrine Hh signaling only in bone marrow-derived mesenchymal stem cell and EMT significantly increased proliferation (10-15%) of TNBC and normal breast cancer cell lines. Addition of adipose-derived mesenchymal stem cells or pharmacological Hh inhibitors (Cyclopamine and Gant61) partially abolished tumor growth indicating that other non-canonical signals are involve in paracrine Hh-driven tumors. Cancer stem cell markers were selectively modulated in co-cultures. Our results suggest that paracrine Hh signaling-driven by bone marrow and EMT mesenchymal are potential therapeutic targets to treating TNBC. Citation Format: Karla P. Ramos, Maribella Domenech. Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in triple negative breast 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 1334. doi:10.1158/1538-7445.AM2017-1334

Adipocyte Accumulation in the Bone Marrow during Obesity and Aging Impairs Stem Cell-Based Hematopoietic and Bone Regeneration

SummaryAging and obesity induce ectopic adipocyte accumulation in bone marrow cavities. This process is thought to impair osteogenic and hematopoietic regeneration. Here we specify the cellular identities of the adipogenic and osteogenic lineages of the bone. While aging impairs the osteogenic lineage, high-fat diet feeding activates expansion of the adipogenic lineage, an effect that is significantly enhanced in aged animals. We further describe a mesenchymal sub-population with stem cell-like characteristics that gives rise to both lineages and, at the same time, acts as a principal component of the hematopoietic niche by promoting competitive repopulation following lethal irradiation. Conversely, bone-resident cells committed to the adipocytic lineage inhibit hematopoiesis and bone healing, potentially by producing excessive amounts of Dipeptidyl peptidase-4, a protease that is a target of diabetes therapies. These studies delineate the molecular identity of the bone-resident adipocytic lineage, and they establish its involvement in age-dependent dysfunction of bone and hematopoietic regeneration.

Synthetic vulnerabilities of mesenchymal subpopulations in pancreatic cancer.

Malignant neoplasms evolve in response to changes in oncogenic signalling. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1-Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α-MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.

Differential epithelial and mesenchymal CTC profiling according to non-small cell lung cancer (NSCLC) molecular subtype.

e23093Background: CTCs may exist as co-operative epithelial and mesenchymal subpopulations or as individual highly plastic cells. Molecular characterisation of NSCLC has revealed it to be a disease...

Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835.

Abstract PR01: Targeting the EMT-immunosuppression loop in lung cancer as a strategy to prevent metastasis

Abstract Either tumor cell epithelial-mesenchymal transition (EMT) or immunosuppression plays the very important role in metastasis during cancer progression. How these distinct processes are co-opted has remained obscure. To address this question, we have generated a cohort of epithelial and mesenchymal cell lines with manipulation of microRNA-200/ZEB1 (an EMT regulatory axis) in KrasLA1/+p53R172HΔg/+ spontaneous mouse model of lung cancer and performed tumor immune cell profiling and functional analysis. The data demonstrates that the microRNA-200 repression and ZEB1 activation target PD-L1 on tumor cells, leading to immunosuppression and metastasis by CD8+ T cell dysfunction, and that marked accumulation of regulatory T cells and myeloid-derived suppressor cells in mesenchymal tumors compared with epithelial tumors. Furthermore, by analyzing two large independent datasets (TCGA and PROSPECT) from lung cancer patients, our study demonstrates that EMT is highly associated with a distinct inflammatory tumor microenvironment, showing the elevation of multiple immune checkpoints such as PD-L1, PD-1, TIM-3, BTLA, CTLA-4 and B7-H3, high levels of tumor-infiltrating Foxp3+ regulatory T cells and immunosuppressive cytokines such as CXCL12, CCL2 and CCL18. The data discovered a link between EMT and immunosuppression, indicating that EMT causes immunosuppression, thereby metastasis. On the other hand, the immunosuppressive microenvironment transforms and/or maintains cancer cells in a mesenchymal state, therefore, cancer cells get the potential/power to metastasize. Immune suppressive cells are able to produce a diverse array of EMT inducers. For instance, T regulatory cells produce TGF-β;, IL-6, IL-10, and TNF-α;. Other immune suppressive cells such as myeloid-derived suppressor cells, tumor-associated macrophages, and tumor-associated neutrophils can also produce strong EMT inducers. Both human and mouse data convincingly established that EMT and immunosuppression form a double positive feed-forward loop, synergizing to promote tumor development. However, when the tumor-bearing mice were treated with anti-PD-L1 antibody, the mesenchymal tumors demonstrated greater sensitivity to the immunotherapy. This is a striking result because we have reported that the mesenchymal subpopulation accounts for aggressive growth, invasion, and metastasis. Since previous studies suggested that some chemotherapeutic agents can induce EMT, we likely find a way, targeting the loop of EMT and immunosuppression, to turn cancer's strengths into targetable weaknesses by combining EMT-induced chemotherapeutics and immunotherapeutics to reduce/prevent tumor metastasis. Citation Format: Limo Chen, Yanyan Lou, John Heymach, Ignacio Wistuba, Stephen Ullrich, Xiao-Feng Qin, Don Gibbons. Targeting the EMT-immunosuppression loop in lung cancer as a strategy to prevent metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR01.

Abstract IA20: Molecular and genomic characterization of invasive circulating tumorcells (iCTCs) from men with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Molecular characterization and genomic analysis of circulating tumor cells (CTCs) may allow for a better understanding of the mechanisms of resistance to therapies in metastatic castration resistant prostate cancer (mCRPC). The Vitatex VitaAssay platform captures invasive CTCs (iCTCs) in a cell surface marker-independent fashion based on their ability to invade a fluorescently-labeled cell adhesion matrix (CAM), allowing for the analysis of multiple CTC subpopulations. We recently demonstrated our ability to detect EMT and stem-like CTC subpopulations with the CAM platform (Friedlander et al., Int J Cancer 2014). We hypothesize that CTC subpopulation diversity is associated with a shorter period of time to resistance for men with mCRPC. Here we sought to estimate epithelial, mesenchymal, stem-like and neuroendocrine iCTC subpopulation diversity in men with CRPC starting abiraterone acetate or enzalutamide therapy and to report the genomic profiles of iCTCs and matched metastatic biopsies. Methods: Blood is being obtained with informed consent from men with mCRPC partaking in either a dose escalation abiraterone trial or the West Coast Dream Team (WCDT) trial. iCTCs are being isolated from the blood samples using the CAM platform coupled with FACS. Paired metastatic biopsies are being obtained as part of the WCDT, when available. For CTC enrichment, iCTCs are defined as CAM+/CD45-/CD14-. For subpopulation characterizations, mesenchymal iCTCs are defined as vimentin+, stem-like iCTCs as CD44+ and neuroendocrine iCTCs as chomogranin (CgA)+. Agilent array comparative genomic hybridization (aCGH) of iCTCs and paired biopsies is being performed. Results: iCTCs were isolated using the CAM platform from 96 men, to date. The average baseline iCTC count per 7.5ml was 160 (range 0-1255). In a subset of 69 subjects, the average baseline CD44+ iCTC count per 7.5ml was 153 (range 0 -1423), and in a subset of 31 patients the median vimentin+ iCTC count per 7.5ml was 116 (range 5-538) and in a subset of 5 patients the median CgA+ iCTC counts per 7.5ml was 79 (range 46 - 185). Using aCGH, copy number aberration is detectable in iCTCs. Some iCTC aCGH profiles resemble paired metastatic biopsy aCGH profiles. Conclusions: Multiple CRPC iCTC subpopulations are identifiable from men with mCRPC, reflecting the heterogeneity of the disease. Data will continue to be collected as part of these two ongoing clinical trials to identify biomarkers of resistance and shed light on resistance pathways. Citation Format: Terence W. Friedlander, Gayatri Premasekharan, Archana Dilip, Jaime Tawney, Vy Ngo, Elizabeth Gilbert, Charles J. Ryan, Eric Small, Pamela L. Paris. Molecular and genomic characterization of invasive circulating tumorcells (iCTCs) from men with metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA20.

Contribution of Leukemia-Induced Alterations in Mesenchymal Cell Subpopulations to Altered Regulation of Leukemic and Normal Stem Cells in the CML BM Microenvironment

Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem cell (HSC) maintenance. The function of microenvironmental niches in regulating leukemia stem cell (LSC) growth is not well understood. We have reported that alterations in chemokine and cytokine expression in the CML BM microenvironment, including reduction in expression of the critical hematopoietic chemokine CXCL12, may differentially alter CML and normal stem cell localization and growth, and provide a competitive growth advantage to CML LSC. Here we evaluated the contribution of leukemia-induced alterations in BM mesenchymal cell subpopulations to altered BM microenvironmental function in CML. Our group has used cell surface markers to characterize mesenchymal cells in the BM of adult mice. These studies have identified three distinct osteoprogenitor subpopulations [CD166+CD146- (CD166+);CD166-CD146+ (CD146+); and CD166-CD146-Sca1+ (Sca1+) cells] as well as a phenotypically defined population of CXCL12-adundant reticular (CAR) cells (Sca1-CD44+CD51+CD106+CD140a+) within CD45-Ter119-CD31- BM mesenchymal cells. We have shown that Sca1+ cells represent primitive mesenchymal progenitors that can generate the other mesenchymal populations, whereas CD146+ cells may represent an intermediate population between Sca1+ cells and CD166+ osteoblasts. Using the SCL-tTA-BCR/ABL mouse model of CML, we examined leukemia-induced alterations in distribution and LTHSC supportive function of mesenchymal cells in CML BM. We observed that the frequency of Sca1+ (p=0.001) and CD146+ (p=0.02) progenitors were significantly increased, and of CD166+ osteoblastic cells (p<0.05) and CAR (p<0.02) cells were significantly reduced, in CML compared to normal BM. CD146+ and CD166+ cells, but not Sca1+ cells, from CML BM showed reduced proliferation in vitro compared to their normal counterparts. To evaluate hematopoietic supportive function, selected normal and CML mesenchymal subpopulations were cocultured with CD45.1 normal and CML LTHSC for 3 days at a 1:1 ratio. LTHSC were then transplanted into CD45.2 recipient mice (200 normal LTHSC/mouse; 1000 CML LTHSC/mouse). Normal LTHSC cocultured with normal Sca1+ (p<0.03), CD146+ (p<0.02), CD166+ (p<0.02), and CAR (p<0.03) cells demonstrated significantly increased hematopoietic engraftment in recipient mice at 16 weeks compared with control LTHSC cultured without mesenchymal cells. In contrast engraftment of normal LTHSC co-cultured with CML compared to normal BM mesenchymal subsets was significantly reduced [Sca1+ (p<0.03), CD146+ (p<0.02), CD166+ (p=0.06), and CAR (p<0.02) cells]. CML LTHSC cultured with normal Sca1+ (p<0.002), CD146+ (p<0.001), CD166+ (p<0.001), and CAR (p<0.03) cells showed significantly increased engraftment in recipient mice after transplantation and increased leukemia development compared with CML LTHSC cultured without stromal cells. CML LTHSC showed similar engraftment in recipient mice after coculture with CML and normal Sca1+ and CD146+ cells, but significantly reduced engraftment after coculture with CML compared to normal CD166+ (p<0.01) and CAR (p<0.02) cells. In conclusion, our studies have identified leukemia associated alterations in the distribution of mesenchymal subpopulations in the CML BM microenvironment, with decrease in more differentiated CD166+ osteoblastic cells and CAR cells and increase in primitive Sca-1+ and CD146+ cells. The reduction in CXCL12 producing CAR cells, and the reduced ability of CML Sca1+ and CD146+ cells to support normal compared to CML LTHSC, may provide a competitive growth advantage to the leukemic clone within the CML BM microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Bcl-xL mediates therapeutic resistance of a mesenchymal breast cancer cell subpopulation.

The transition from an epithelial to a mesenchymal phenotype (EMT) confers increased invasiveness and clonogenic potential to tumor cells. We used a breast epithelium-derived cell culture model to evaluate the impact of EMT on the cellular sensitivity towards chemotherapeutics and apoptotic stimuli. Cells that had passed through an EMT acquired resistance towards chemotherapeutics and death ligands. Mechanistically, we found that the levels of the apoptosis inhibitor Bcl-xL were strongly enhanced in mesenchymal versus epithelial cells, whereas the pro-apoptotic proteins Bim and Puma were diminished. Clinical samples from breast cancer showed enhanced Bcl-xL staining in cells that had dispersed into the desmoplastic stroma, as compared to cells that were part of large tumor cell aggregates, suggesting increased Bcl-xL expression when cells invade the stroma. Bcl-xL was necessary for apoptotic resistance in mesenchymal cells, and its expression was sufficient to confer such resistance to epithelial cells. To antagonize Bcl-xL, BH3-mimetics were used. They successfully interfered with the proliferation and survival of mesenchymal cells, and also inhibited the growth of xenograft tumors raised from the mesenchymal subpopulation. We conclude that enhanced Bcl-xL levels confer resistance to cells upon EMT, and that Bcl-xL represents a promising target for therapy directed against invasive cancer cells.

Controlling the dynamics of cell transition in heterogeneous cultures using surface chemistry.

Developing materials that can preferentially select defined cancer cell populations for biological characterization will greatly enhance our understanding of cancer cell growth, differentiation, and invasion. The transitional events between epithelial and mesenchymal phenotypes are particularly crucial, as primary tumors and secondary metastasis are generally epithelial in nature, whereas circulating mesenchymal cells derived from primary epithelial cells appear to facilitate the spread of disease and its resistance to therapy. This study describes an amino-functionalized material, which promotes the enrichment of an epithelial phenotype from a single cell line containing both epithelial and mesenchymal subpopulations of cancer cells. The isolation and transitional control of such subpopulations using functional materials will advance understanding of the disease process, have a significant impact on the downstream development of new targeted cancer therapeutics, and also be applicable to tissue engineering and regenerative medicine.

α6-Integrin Is Required for the Adhesion and Vasculogenic Potential of Hemangioma Stem Cells

Infantile hemangioma (IH) is the most common tumor of infancy. Hemangioma stem cells (HemSC) are a mesenchymal subpopulation isolated from IH CD133+ cells. HemSC can differentiate into endothelial and pericyte/smooth muscle cells and form vascular networks when injected in immune-deficient mice. α6-Integrin subunit has been implicated in the tumorgenicity of glioblastoma stem cells and the homing properties of hematopoietic, endothelial, and mesenchymal progenitor cells. Therefore, we investigated the possible function(s) of α6-integrin in HemSC. We documented α6-integrin expression in IH tumor specimens and HemSC by RT-qPCR and flow cytometry. We examined the effect of blocking or silencing α6-integrin on the adhesive and proliferative properties of HemSC in vitro and the vasculogenic and homing properties of HemSC in vivo. Targeting α6-integrin in cultured HemSC inhibited adhesion to laminin but had no effect on proliferation. Vessel-forming ability in Matrigel implants and hepatic homing after i.v. delivery were significantly decreased in α6-integrin siRNA-transfected HemSC. In conclusion, α6-integrin is required for HemSC adherence to laminin, vessel formation in vivo, and for homing to the liver. Thus, we uncovered an important role for α6 integrin in the vasculogenic properties of HemSC. Our results suggest that α6-integrin expression on HemSC could be a new target for antihemangioma therapy.

Molecular and genomic characterization of invasive circulating tumor cells (iCTCs) from men with metastatic castration-resistant prostate cancer (mCRPC).

120 Background: Identification, enumeration, and genomic analysis of circulating tumor cells (CTCs) may allow for a better understanding of the mechanisms of resistance to therapies in metastatic castration-resistant prostate cancer (mCRPC). The Vitatex VitaAssay platform captures invasive CTCs (iCTCs) in a cell surface marker-independent fashion based on their ability to invade a fluorescently-labeled cell-adhesion matrix (CAM), allowing for the analysis of multiple CTC subpopulations. Here we sought to estimate epithelial, mesenchymal, and stem-like iCTC subpopulation diversity in men with CRPC starting abiraterone acetate therapy, to compare the genomic profiles of iCTCs to matched metastatic biopsies, and to explore the potential for 2D and 3D CTC culture. Methods: iCTCs were isolated from men with mCRPC using the CAM platform, and paired metastatic biopsies were performed. iCTCs were defined as CAM+/CD45-/CD14-/DAPI+, mesenchymal iCTCs as vimentin+/CAM+/CD45-/CD14-/DAPI+, and stem-like iCTCs as CD44+/CAM+/CD45-/CD14-/DAPI+. iCTCs were enumerated and purified using FACS. Agilent array comparative genomic hybridization (aCGH) of iCTCs and paired biopsies was performed, and to explore the potential for ex-vivo cell expansion and spheroid formation, iCTCs were cultured separately in CAM and in matrigel for up to 10 days. Results: iCTCs were isolated using the CAM platform from 29 men, of whom seven have undergone paired metastatic biopsy. The median pre-FACS purity was 1.06% (range 0.11%-10.16%). Post-FACS purity was increased to greater than 90%, and a median of 60 (range 2 to 1,314) iCTCs/7.5ml were detected by FACS. Both vimentin+ and CD44+ iCTCs are detectable, and compromise between 10 to 50% of total iCTCs. iCTC aCGH profiles resemble paired soft tissue biopsy, in vitro iCTCs culture is feasible, and iCTC spheroids were observed. Conclusions: Multiple CRPC iCTC subpopulations are identifiable from men starting abiraterone therapy, and cell sorting techniques increase iCTC purity. iCTCs resemble metastatic CRPC tissue and can be expanded in culture. Further enumeration, genomic profiling, and clinical correlation of paired iCTCs taken from men with abiraterone-resistant CRPC is underway, and may shed light on mechanisms of abiraterone resistance.

A central role for vimentin in regulating repair function during healing of the lens epithelium

Mock cataract surgery provides a unique ex vivo model for studying wound repair in a clinically relevant setting. Here wound healing involves a classical collective migration of the lens epithelium, directed at the leading edge by an innate mesenchymal subpopulation of vimentin-rich repair cells. We report that vimentin is essential to the function of repair cells as the directors of the wound-healing process. Vimentin and not actin filaments are the predominant cytoskeletal elements in the lamellipodial extensions of the repair cells at the wound edge. These vimentin filaments link to paxillin-containing focal adhesions at the lamellipodial tips. Microtubules are involved in the extension of vimentin filaments in repair cells, the elaboration of vimentin-rich protrusions, and wound closure. The requirement for vimentin in repair cell function is revealed by both small interfering RNA vimentin knockdown and exposure to the vimentin-targeted drug withaferin A. Perturbation of vimentin impairs repair cell function and wound closure. Coimmunoprecipitation analysis reveals for the first time that myosin IIB is associated with vimentin, linking vimentin function in cell migration to myosin II motor proteins. These studies reveal a critical role for vimentin in repair cell function in regulating the collective movement of the epithelium in response to wounding.

Comparative Secretome Analysis of Epithelial and Mesenchymal Subpopulations of Head and Neck Squamous Cell Carcinoma Identifies S100A4 as a Potential Therapeutic Target

Epithelial-mesenchymal transition (EMT) is a key contributor in tumor progression and metastasis. EMT produces cellular heterogeneity within head and neck squamous cell carcinomas (HNSCC) by creating a phenotypically distinct mesenchymal subpopulation that is resistant to conventional therapies. In this study, we systematically characterized differences in the secretomes of E-cadherin high epithelial-like and E-cadherin low mesenchymal-like subpopulations using unbiased and targeted proteomics. A total 1765 proteins showed significant changes with 177 elevated in the epithelial subpopulation and 173 elevated in the mesenchymal cells. Key nodes in affected networks included NFκB, Akt, and ERK, and most implicated cellular components involved various aspects of the extracellular matrix. In particular, large changes were observed in multiple collagens with most affected collagens at much higher abundance levels in the mesenchymal subpopulation. These cells also exhibited a secretome profile resembling that of cancer-associated fibroblastic cells (CAF). S100A4, a commonly used marker for cancer-associated fibroblastic cells, was elevated more than 20-fold in the mesenchymal cells and this increase was further verified at the transcriptome level. S100A4 is a known mediator of EMT, leading to metastasis and EMT has been proposed as a potential source of cancer-associated fibroblastic cells in solid tumors. S100A4 knockdown by small interfering RNA led to decreased expression, secretion and activity of matrix metalloproteinase 2, as verified by quantitative PCR, multiple reaction monitoring and zymography analyses, and reduced invasion in collagen-embedded spheroids. Further confirmation in three-dimensional organotypic reconstructs showed less invasion and advanced differentiation in the S100A4 RNA interference samples. Orthotopic metastasis model, developed to validate the findings in vivo, demonstrated a decrease in spontaneous metastasis and augmented differentiation in the primary tumor in siS100A4 xenografts. These results demonstrate the value of secretome profiling to evaluate phenotypic conversion and identify potential novel therapeutic targets such as S100A4.