Label Retention Research Articles

What is a stem cell?

The historical roots of the stem cell concept are traced with respect to its usage in embryology and in hematology. The modern consensus definition of stem cells, comprising both pluripotent stem cells in culture and tissue-specific stem cells in vivo, is explained and explored. Methods for identifying stem cells are discussed with respect to cell surface markers, telomerase, label retention and transplantability, and properties of the stem cell niche are explored. The CreER method for identifying stem cells in vivo is explained, as is evidence in favor of a stochastic rather than an obligate asymmetric form of cell division. In conclusion, it is found that stem cells do not possess any unique and specific molecular markers; and stem cell behavior depends on the environment of the cell as well as the stem cell's intrinsic qualities. Furthermore, the stochastic mode of division implies that stem cell behavior is a property of a cell population not of an individual cell. In this sense, stem cells do not exist in isolation but only as a part of multicellular system. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Methods and Principles Adult Stem Cells, Tissue Renewal, and Regeneration > Environmental Control of Stem Cells.

Rebooting the Zahnreihen: Analysis of the Jaw‐wide Patterning cues in the Adult Gecko Dentition

Unlike mammals, it is common for reptiles to replace their teeth throughout life. Through exploring the mechanisms of tooth replacement, particularly in adult reptiles we will learn about the cells and molecules that are required for the regeneration of the dentition. Wholemount preparations, x-rays and μCT scans of most lizards show that each functional tooth appears to line up with a series of unerupted teeth that are in progressively less mature stages of development going posteriorly (Zahnreihen). The position of the Zahnreihen is at approximately 147° to the jaw margin. The next teeth that are fated to replace a functional tooth do not appear in the mouth until they have reached a threshold size and have moved into position. It has been hypothesized that the unerupted teeth secrete factors that regulate the pace of tooth formation and spacing. All unerupted teeth are connected to each other via a dental lamina that extends around the jaw, extending deep into the soft tissue lingual to the functional teeth. Using an unbiased, surgical approach we asked whether there were signals emanating from the unerupted teeth and dental lamina. We carried out 4 types of surgeries in defined regions: enucleation of unerupted teeth with minimal disruption of the dental lamina, enucleation plus scrambling of the dental lamina, enucleation plus chemical cauterization of remaining epithelium or a sham control. Animals were analyzed at 7d, 14d, or 1–8 months. The macroscopic and microscopic phenotypes confirmed that teeth were removed successfully and that recovery was taking place except when cauterization was carried out. There was an initial wave of apoptosis that tapered off by 5 days. Next a spike in proliferation occurred in both torn epithelium and surrounding mesenchyme that lasted for at least 1 week. In long term followup we found that simple tooth removal delays but does not impact overall patterning. In contrast, cauterization of the dental lamina causes a block in tooth formation that does not recover from the cut edges (up to 8 months follow-up). The mesenchyme on its own is completely unable to induce new teeth from other types of epithelia such as oral mucosa or glandular epithelium. The dental epithelium is therefore instructive and is the key tissue that must be present for new teeth to form. Molecular readouts that will be presented include the location of putative stem cells (BrdU label retention, SOX2 antibody staining), changes in gene expression (in situs and RNAseq) and evidence of polarity of the epithelium being re-established (in situs). This data will be used to map out the sequence of recovery in dental epithelium leading to reestablishment of the Zahnreihen. Support or Funding Information Studies were supported by an NSERC Discovery Grant (RGPIN-2016-05477) to JMR. TMG was supported by an NIH F32 Postdoctoral Fellowship (F32DE024948). KB is supported by Killam and Michael Smith Foundation for Health Research postdoctoral fellowships. Palatal view of an adult gecko with approximately 40 functional teeth in each quadrant. Each marginal tooth defines the start of a Zahnreihen (diagonal row of unerupted teeth). Not shown is the continuous dental lamina to which all teeth are attached. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Establishing and characterizing lacrispheres from human lacrimal gland for potential clinical application.

Lacrimal gland (LG) dysfunction leading to dry eye syndrome (DES) is an important cause of ocular morbidity. One of the potential and promising long-term management therapies for restoration of LG function could be transplantation of autologous ex vivo expanded stem cells. The present study was aimed at exploring the 2D and 3D cultures of human LG, identifying inherent stem cells and evaluating their secretory potential. Fresh human lacrimal gland (HuLG) (n = 5) from patients undergoing therapeutic exenteration was harvested after ethical approval and informed consent. The gland was enzymatically digested and the isolated cells plated in Hepato-STIM media supplemented with l-glutamine, epidermal growth factor, fibroblast growth factor, and N-2 supplement. The native HuLG and the cultured spheres (DIV14-16) were evaluated for presence of stem cells (CD117 expression, quiescence, BrdU label retention, cell cycle, colony forming efficiency) and differentiation (secretion of tear proteins). Under the established culture conditions, suspension 3D cultures of human "lacrispheres" could be maintained and propagated for 3-4weeks. The spheres consist of both acinar as well as ductal cells with evidence of stem cells (0.8 ± 0.05% CD117+ cells), BrdU label retention (9.31 ± 0.41%), G0/G1 profile similar to native lacrimal cells at isolation (76.9 versus 79.9%) and colony forming units (3.1%). The lacrispheres also secreted quantifiable levels of tear proteins (lysozyme, lactoferrin, scIgA) into the conditioned media. The study provides promising, first-of-its-kind evidence for the generation of lacrispheres from fresh HuLG, with enriched population of stem cells and secretory competent differentiated cells. The dual properties of these spheres make them a highly suitable source of transplantable cells for restoring the structure and function of damaged lacrimal gland.

Site-Selective Deuteration of N-Heterocycles via Iridium-Catalyzed Hydrogen Isotope Exchange

The application of iridium(I) NHC/phosphine catalysts has delivered highly selective deuteration of indole, azaindole, and pyrrole N-heterocycles, which represent an important and relatively underexplored class of labeling substrates. Common N-protecting groups have been used to selectively direct C–H activation and can be removed under mild conditions with retention of the deuterium label. The method is exemplified by the labeling of the drug molecule sumatriptan. Complementary DFT studies have been conducted to facilitate the rationalization of the very good selectivity offered by the mild and convenient labeling process.

Radionuclide Tumor Targeting Using ADAPT Scaffold Proteins: Aspects of Label Positioning and Residualizing Properties of the Label.

Visualization of cancer-associated alterations of molecular phenotype using radionuclide imaging is a noninvasive approach to stratifying patients for targeted therapies. The engineered albumin-binding domain-derived affinity protein (ADAPT) is a promising tracer for radionuclide molecular imaging because of its small size (6.5 kDa), which satisfies the precondition for efficient tumor penetration and rapid clearance. Previous studies demonstrated that the human epidermal growth factor receptor type 2 (HER2)-targeting ADAPT6 labeled with radiometals at the N terminus is able to image HER2 expression in xenografts a few hours after injection. The aim of this study was to evaluate whether the use of a nonresidualizing label or placement of the labels at the C terminus would further improve the targeting properties of ADAPT6. Methods: Two constructs, Cys2-ADAPT6 and Cys59-ADAPT6, having the (HE)3DANS sequence at the N terminus were produced and site-specifically labeled using 111In-DOTA or 125I-iodo-((4-hydroxyphenyl)ethyl) maleimide (HPEM). The conjugates were compared in vitro and in vivo. HER2-targeting properties and biodistribution were evaluated in BALB/C nu/nu mice bearing ovarian carcinoma cell (SKOV-3) xenografts. Results: Specific HER2 binding and high affinity were preserved after labeling. Both Cys2-ADAPT6 and Cys59-ADAPT6 were internalized slowly by HER2-expressing cancer cells. Depending on the label position, uptake at 4 h after injection varied from 10% to 22% of the injected dose per gram of tumor tissue. Regardless of terminus position, the 125I-HPEM label provided more than 140-fold lower renal uptake than the 111In-DOTA label at 4 after injection. The tumor-to-organ ratios were, in contrast, higher for both of the 111In-DOTA-labeled ADAPT variants in other organs. Tumor-to-blood ratios for 111In-labeled Cys2-ADAPT6 and Cys59-ADAPT6 did not differ significantly (250-280), but 111In-DOTA-Cys59-ADAPT6 provided significantly higher tumor-to-lung, tumor-to-liver, tumor-to-spleen, and tumor-to-muscle ratios. Radioiodinated variants had similar tumor-to-organ ratios, but 125I-HPEM-Cys59-ADAPT6 had significantly higher tumor uptake and a higher tumor-to-kidney ratio. Conclusion: Residualizing properties of the label strongly influence the targeting properties of ADAPT6. The position of the radiolabel influences targeting as well, although to a lesser extent. Placement of a label at the C terminus yields the best biodistribution features for both radiometal and radiohalogen labels. Low renal retention of the radioiodine label creates a precondition for radionuclide therapy using 131I-labeled HPEM-Cys59-ADAPT6.

Sca-1 expression level identifies quiescent hematopoietic stem and progenitor cells

Blood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC) and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem cell antigen 1 (SCA-1/LY6A). Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi) identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage-SCA-1+KIT+ (LSK) population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN). We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.

Regulatory elements driving the expression of skeletal lineage reporters differ during bone development and adulthood

To improve bone healing or regeneration more insight in the fate and role of the different skeletal cell types is required. Mouse models for fate mapping and lineage tracing of skeletal cells, using stage-specific promoters, have advanced our understanding of bone development, a process that is largely recapitulated during bone repair. However, validation of these models is often only performed during development, whereas proof of the activity and specificity of the used promoters during the bone regenerative process is limited. Here, we show that the regulatory elements of the 6kb collagen type II promoter are not adequate to drive gene expression during bone repair. Similarly, the 2.3kb promoter of collagen type I lacks activity in adult mice, but the 3.2kb promoter is suitable. Furthermore, Cre-mediated fate mapping allows the visualization of progeny, but this label retention may hinder to distinguish these cells from ones with active expression of the marker at later time points. Together, our results show that the lineage-specific regulatory elements driving gene expression during bone development differ from those required later in life and during bone repair, and justify validation of lineage-specific cell tracing and gene silencing strategies during fracture healing and bone regenerative applications.

Quiescent Oct4+ Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein+ NSCs in the Adult Mouse Brain.

Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP)- Oct4+ cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP+ NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4fl/fl ;Sox1Cre (Oct4CKO ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP+ dNSCs, in these Oct4CKO mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine β-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP+ dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082.

Abstract 1925: Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance

Abstract Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy because they are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting. Citation Format: Ashley Gilbert, Kiera Walker, Anh Tran, Yancey Gillespie, Raj Singh, Anita B. Hjelmeland. Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance [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 1925. doi:10.1158/1538-7445.AM2017-1925

Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers

In order to identify reliable markers of corneal epithelial stem cells, we employed an inducible transgenic “pulse-chase” murine model (K5Tta × TRE-H2BGFP) to localize, purify, and characterize slow cycling cells in the cornea. The retention of GFP labeling in slowly dividing cells allowed for localization of these cells to the corneal limbus and their subsequent purification by FACS. Transcriptome analysis from slow cycling cells identified differentially expressed genes when comparing to GFP- faster-dividing cells. RNA-Seq data from corneal epithelium were compared to epidermal hair follicle stem cell RNA-Seq to identify genes representing common putative stem cell markers or determinants, which included Sox9, Fzd7, Actn1, Anxa3 and Krt17. Overlapping retention of GFP and immunohistochemical expression of Krt15, ΔNp63, Sox9, Actn1, Fzd7 and Krt17 were observed in our transgenic model. Our analysis presents an array of novel genes as putative corneal stem cell markers.

SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem and Progenitor Cells.

SummaryBlood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC) and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem cell antigen 1 (SCA-1/LY6A). Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi) identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage−SCA-1+KIT+ (LSK) population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN). We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.

Label retention and stem cell marker expression in the developing and adult prostate identifies basal and luminal epithelial stem cell subpopulations

BackgroundProstate cancer is the second most frequent cancer among males worldwide, and most patients with metastatic disease eventually develop therapy-resistant disease. Recent research has suggested the existence of cancer stem-like cells, and that such cells are behind the therapy resistance and progression.MethodsHere, we have taken advantage of the relatively quiescent nature of stem cells to identify the slow-cycling label-retaining stem cell (LRC) populations of the prostate gland. Mice were pulsed with bromodeoxyuridine (BrdU) during prostate organogenesis, and the LRC populations were then identified and characterized in 5-day-old and in 6-month-old adult animals using immunohistochemistry and immunofluorescence.ResultsQuantification of LRCs in the adult mouse prostate showed that epithelial LRCs were significantly more numerous in prostatic ducts (3.7 ± 0.47% SD) when compared to the proximal (1.4 ± 0.83%) and distal epithelium (0.48 ± 0.08%) of the secretory lobes. LRCs were identified in both the basal and epithelial cell layers of the prostate, and LRCs co-expressed several candidate stem cell markers in a developmental and duct/acini-specific manner, including Sca-1, TROP-2, CD133, CD44, c-kit, and the novel prostate progenitor marker cytokeratin-7. Importantly, a significant proportion of LRCs were localized in the luminal cell layer, the majority in ducts and the proximal prostate, that co-expressed high levels of androgen receptor in the adult prostate.ConclusionsOur results suggest that there are separate basal and luminal stem cell populations in the prostate, and they open up the possibility that androgen receptor-expressing luminal stem-like cells could function as cancer-initiating and relapse-responsible cells in prostate cancer.

Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays

In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g., lactate dehydrogenase, or measurement of released exogenous labels, e.g., 51Cr, europium or calcein. ADCC assays based on the detection of exogenous labels released from lysed target cells generally show higher sensitivity and require shorter incubation times. However, target cells are usually labeled immediately prior to assay, which inadvertently introduces additional assay variations due to differences in target cell conditions and labeling/handling processes. In this report, we describe the use of thaw-and-use pre-labeled target cells for ADCC assays. Thaw-and-use target cells in our experiments were pre-labeled with the fluorescent dye calcein AM, cryopreserved in single-use aliquots and used directly in assays after thawing. Upon thaw, the pre-labeled cells displayed viability and label retention comparable to freshly labeled cells, responded to ADCC mediated by both peripheral blood mononuclear cells and engineered natural killer cells, performed stably for at least 3 years and provided favorable precision and accuracy to ADCC assays. Implementation of thaw-and-use pre-labeled target cells in ADCC assays can help to alleviate both cell culture and dye labeling derived variability, increase the flexibility of assay scheduling and improve assay consistency and robustness.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Modeling Physiologic Microenvironments in Three-Dimensional Microtumors Maintains Brain Tumor Initiating Cells.

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity, including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy, because BTICs are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using the human-derived biomatrix HuBiogel™ and maintained with physiologically relevant microenvironments. BITC percentages were determined using cell surface marker expression, label retention, and neurosphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient-restricted microtumors significantly increased the percentage of both CD133+ and CFSEhigh cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintained neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation.

To avoid xenogeneic infection, we report a novel protocol for producing animal-derived component-free oral mucosal epithelial cells (OMECs) sheet for transplantation, in which collagenase was used to replace dispase II/trypsin-EDTA for digesting oral mucosal tissue, and human platelet-derived PLTMax to replace fetal bovine serum. The resulting epithelial aggregates were expanded on de-epithelialized amniotic membranes without 3T3 feeder cells, and serum-free EpiLife was used to reduce contamination by submucosal mesenchymal cells. The OMEC sheets thus generated showed similar positive keratin 3/76-positive and keratin 8-negative staining patterns compared with those generated by the original protocol. Colony formation efficiency assay, BrdU label retention assay, and p63 and p75NTR immunostaining results indicated that higher proliferative potentials and more progenitor cells were preserved by the modified protocol. TaqMan array analysis revealed that the transcription of integrin-linked kinase (ILK) was up-regulated along with an increase in β-catenin signaling and its downstream cell cycle modulators, cyclin D1 and p27KIP1. Furthermore, ILK silencing led to the inhibition of nuclear β-catenin accumulation, suppressed p63 expression, and reduced the expression of cyclin D1 and p27KIP1; these observations suggest that ILK/β-catenin pathway may be involved in cell proliferation regulation during the ex vivo expansion of OMECs for transplantation purposes.

TMOD-18. THREE-DIMENSIONAL MICROTUMORS IN PHYSIOLOGIC MICROENVIRONMENTS MAINTAIN BRAIN TUMOR INITIATING CELLS

Elucidation of mechanisms of brain tumor initiating cell (BTIC) maintenance and therapeutic resistance offers great promise for development of novel anti-tumor treatments. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined if three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were then determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. For example, fifteen percent of all brain tumor cells from a xenograft propagated as microtumors were CD133+, whereas only one percent of neurosphere cultured cells expressed the BTIC marker. Relative to CD133, CFSE was retained in a higher percentage of cells, but CFSE+ cells were still increased in the microtumor condition in comparison to neurospheres. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

Abstract 4394: Phenotypic heterogeneity of disseminated tumor cells is predetermined by primary tumor hypoxic microenvironments

Abstract Heterogeneity of primary tumors (PTs) is reflected by genetic and epigenetic diversity, as well as diverse PT microenvironments. Whether PT microenvironments might influence the fate of disseminating tumor cells (DTC) has never been explored in situ. We previously defined, in breast cancer, a dormancy signature (DS) associated with longer metastasis-free periods. Key genes in the DS induce quiescence and are also regulated by hypoxia. Interestingly, a main response of tumor cells to hypoxia is growth arrest, while clinical evidence links hypoxic tumors to increased therapy resistance and a worse outcome. We hypothesized that hypoxic PT microenvironments may spawn a subpopulation of DTCs that, by virtue of becoming dormant, might escape therapies and eventually fuel incurable metastasis. We used H2B-GFP inducible HEp3 HNSCC and photo-switchable H2B-Dendra2 (green-to-red fluorescence) expressing MDA-MB-231 and ZR-75-1 human breast cancer cell lines to identify cells from hypoxic microenvironments. To initiate spatially defined hypoxic microenvironments in vivo in primary tumors we implanted induction NANo IntraVItal Devices (iNANIVIDs) carrying a hypoxia-mimetic agent (desferrioxamine - DFOM) in T-HEp3 tumors or exposed cultured MDA-MB-231 or ZR-75-1 cells in vitro to 21% or 1% O2. The regions influenced by the DFOM-iNANIVID displayed significant upregulation of p27, NR2F1 and DEC2 (dormancy genes), as well as induction of hypoxia markers (GLUT1, HIF1α). Human HNSCC PT samples showed the same link between spontaneous hypoxic regions and upregulation of dormancy markers. We found a significant increase in quiescent lung DTCs of hypoxia induced T-HEp3 or MDA-MB-231 cells, traceable >2 weeks after extravasation by fluorescent label retention. Significantly more single, non-proliferating HEp3 DTCs originating from the iNANIVID induced hypoxic regions showed a dormant profile compared to DTCs originating from a normoxic milieu. Only the hypoxic pre-treated group was able to form micrometastases at 10 days after injection, suggesting the presence of a more aggressive sub clone in this group. Analysis in 3D culture models revealed that ZR-75-1 cells (ER+) were more prone to enter a prolonged quiescent state after exposure to hypoxia (1% O2) while this response was not observed in MDA-MB-231 (TN). The induction of quiescence in ZR-75-1 was NR2F1 dependent. Lastly, using a spontaneously metastatic PyMT driven Dendra2-tagged breast cancer model in immunocompetent mice, we found that ∼75% of dormant DTCs upregulate the dormancy marker NR2F1 at or soon after reaching the lung, suggesting a rapid induction of dormancy upon reaching target organs. We propose that hypoxic PT microenvironments increase phenotypic heterogeneity of DTCs and lead to the expression of the DS. These DTCs may be more prone to enter dormancy, evade anti-proliferative therapies and eventually fuel metastasis. Citation Format: Georg Fluegen, Alvaro Avivar-Valderas, Yarong Wang, Michael R. Padgen, James K. Williams, Vladislav V. Verkhusha, David Entenberg, Kevin W. Eliceiri, James Castracane, Patricia J. Keely, John S. Condeelis, Julio A. Aguirre-Ghiso. Phenotypic heterogeneity of disseminated tumor cells is predetermined by primary tumor hypoxic microenvironments. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4394.

Mouse Label-Retaining Cells Are Molecularly and Functionally Distinct From Reserve Intestinal Stem Cells

Intestinal homeostasis and regeneration after injury are controlled by 2 different types of cells: slow cycling, injury-resistant reserve intestinal stem cells (ISCs) and actively proliferative ISCs. Putative reserve ISCs have been identified using a variety of methods, including CreER insertions at Hopx or Bmi1 loci in mice and DNA label retention. Label-retaining cells (LRCs) include dormant stem cells in several tissues; in the intestine, LRCs appear to share some properties with reserve ISCs, which can be marked by reporter alleles. We investigated the relationships between these populations. Studies were performed in Lgr5-EGFP-IRESCreERT2, Bmi1-CreERT2, Hopx-CreERT2, and TRE-H2BGFP::Hopx-CreERT2::lox-stop-lox-tdTomato mice. Intestinal epithelial cell populations were purified; we compared reporter allele-marked reserve ISCs and several LRC populations (marked by H2B-GFP retention) using histologic flow cytometry and functional and single-cell gene expression assays. LRCs were dynamic and their cellular composition changed with time. Short-term LRCs had properties of secretory progenitor cells undergoing commitment to the Paneth or enteroendocrine lineages, while retaining some stem cell activity. Long-term LRCs lost stem cell activity and were a homogenous population of terminally differentiated Paneth cells. Reserve ISCs marked with HopxCreER were primarily quiescent (in G0), with inactive Wnt signaling and robust stem cell activity. In contrast, most LRCs were in G1 arrest and expressed genes that are regulated by the Wnt pathway or are in the secretory lineage. LRCs are molecularly and functionally distinct from reporter-marked reserve ISCs. This information provides an important basis for future studies of relationships among ISC populations.

Abstract A43: Phenotypic heterogeneity of disseminated tumor cells is predetermined by primary tumor hypoxic microenvironments

Abstract Heterogeneity within and between primary tumors (PTs) is reflected by genetic and epigenetic diversity and varying PT microenvironments. Further, whether PT microenvironments might influence the fate of disseminating tumor cells (DTC) has never been explored. We found that breast tumors enriched for a specific dormancy signature (DS+) displayed longer metastasis-free periods than those poor (DS-) for the signature. Key genes in the DS induce quiescence and are also regulated by hypoxia. Clinical evidence links hypoxic tumors to increased therapy resistance and a worse outcome. However, a main response of tumor cells to hypoxia is growth arrest, but how this response is linked to the clinical outcome is unknown. We hypothesized that hypoxic PT microenvironments may spawn a subpopulation of DTCs that, by virtue of becoming dormant, might escape therapies and eventually fuel incurable metastasis. We used H2B-GFP inducible HEp3 HNSCC and photo-switchable (green-to-red fluorescence) H2B-Dendra2 expressing MDA-MB-231 and ZR-75-1 human breast cancer cell lines to identify cells from hypoxic microenvironments. To initiate spatially defined hypoxic microenvironments in primary tumors we implanted induction NANo IntraVItal Devices (iNANIVIDs) carrying a hypoxia-mimetic agent (desferrioxamine - DFOM) in T-HEp3 tumors in vivo or exposed cultured MDA-MB-231 or ZR-75-1 cells in vitro to either 21% or 1% O2. The regions influenced by the DFOM-iNANIVID displayed significant upregulation of p27, NR2F1 and DEC2 (dormancy genes), as well as induction of hypoxia markers (GLUT1, HIF1α). Human HNSCC PT samples showed the same link between spontaneous hypoxic regions and up regulation of dormancy markers. We further found a significant increase in quiescent lung DTCs of hypoxia induced H2B-GFP T-HEp3 or H2B-Dendra2 MDA-MB-231 cells, traceable > 2 weeks after extravasation using H2B-GFP and H2B-Dendra2-RED label retention. Using human Vimentin to screen for HEp3 tumor cells in lungs, we found that single, unproliferating DTCs originating from the iNANIVID induced hypoxic regions showed a dormant profile (upregulation of p27, NR2F1, DEC2 and TGFβ2) compared to DTCs originating from a normoxic milieu. Simultaneously, only the hypoxic pre-treated group was able to form micro-metastasis at 10 days after injection, suggesting the presence of a more aggressive sub clone in this group. Further, analysis in 3D culture models revealed that ER+/DS+ breast cancer cells (ZR-75-1) are more prone to enter a prolonged quiescent state after a brief exposure to hypoxia (1% O2) in an NR2F1-dependent manner. This response is not observed in triple negative/DS- breast cancer cells. Lastly, using a spontaneously metastatic PyMT driven Dendra2-tagged breast cancer model, we found that ~75% of dormant DTCs up regulate the dormancy marker NR2F1 at or soon after reaching the lung, suggesting a rapid induction of dormancy upon reaching target organs. We propose that hypoxic primary tumor stress microenvironments increase phenotypic heterogeneity of DTCs and leads to the expression of the DS. Upon spreading, these DTCs may be more prone to enter dormancy, evade anti-proliferative therapies and eventually fuel metastasis. Citation Format: Georg Fluegen, Alvaro Avivar-Valderas, Yarong Wang, Michael R. Padgen, James K. Williams, Vladislav Verkhusha, Julie F. Cheung, David Entenberg, James Castracane, Patricia J. Keely, John Condeelis, Julio A. Aguirre-Ghiso. Phenotypic heterogeneity of disseminated tumor cells is predetermined by primary tumor hypoxic microenvironments. [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 A43.