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Keywords:

  • polysialic acid;
  • NCAM;
  • small-cell lung cancer;
  • cell adhesion;
  • glycoslylation;
  • extracellular matrix;
  • antibody mimetic;
  • flow cytometry

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. Literature Cited
  8. Supporting Information

Polysialylation of neural cell adhesion molecule (NCAM) in small-cell lung cancer (SCLC) is thought to regulate NCAM-mediated cell–surface interactions, imparting antiadhesive properties to cells. However, SCLC cells in culture demonstrate anchorage-independent growth and spontaneously generate adherent forms. Here, the ability of polySia-NCAM to influence cell proliferation and adherence is unclear. We analyzed live SCLC cell polySia-NCAM expression by flow cytometry, using the novel combination of a polySia antibody-mimetic eGFP-tagged endosialidase and the viability dye DRAQ7. Enrichment for adherence (<30 population doublings) in SCLC cell lines resolved populations with increased (SHP-77 and COR-L279) or negligible (NCI-H69) polysialylation compared with nonadherent parent populations. Adherent forms retained NCAM expression as confirmed by immunofluorescence and immunoblotting. Initial transition to adherence and loss of polysialylation in NCI-H69 was linked to a reduced proliferation rate with no increase in cell death. This reduced proliferation rate was reiterated in vivo as determined by the growth of noninvasive subcutaneous xenografts in mice. Continued selection for enhanced substrate adherence in NCI-H69 (>150 population doublings) resolved cells with stable re-expression of polySia and increased growth rates both in vitro and in vivo. Endoneuraminidase removal of polySia from re-expressing cells showed that rapid adherence to extracellular matrix components was functionally independent of polySia. PolySia expression was not altered when isolated adherent forms underwent enforced cell–cell contact in three-dimensional culture. Coculture of polySia expression variants modulated overall polySia expression profiles indicating an influence of SCLC microcommunity composition independent of substrate adherence potential. We conclude that an obligatory linkage between substrate adherence potential and polySia expression is rejected for SCLC cells. We suggest that a degree of homeostasis operates to regulate polysialylation within heterogeneous cell populations. The findings suggest a new model for SCLC progression while the application of live cell profiling of polysialylation could be used to assess polySia-NCAM-targeted therapies. © 2013 International Society for Advancement of Cytometry

Small-cell lung cancer (SCLC) is an aggressive disease with early metastasis and a propensity for chemoresistance [1]. SCLC proliferation is supported by autocrine loops through the expression of neuropeptides and their receptors [1], suggesting that microcommunity interactions may be crucial in SCLC biology. Constitutive JAK2/STAT3 activation drives anchorage-independent in vitro growth [1], consistent with the tendency of SCLC cells to grow as cell aggregates, without substrate attachment but with varying degrees of cell-cell contact [1]. This nonadherent, spheroid-like growth form shown by classic SCLC lines has generated technical problems for study and inclusion into screening systems [1]. Interestingly, nonadherent “classical” SCLC cell populations can spontaneously and apparently stochastically generate adherent forms [6-9]. Such adherence transitions are poorly understood and may be occult in the absence of substrate availability. They may also appear to be infrequent if proliferation is compromised. These heterogeneous SCLC microcommunities frequently harbor nonviable cells and present a challenge for efficient dissociation with surface marker preservation and live-cell analysis by flow cytometry.

Adherence transitions have the potential to promote chemoresistance via enhanced interactions with extracellular matrix (ECM) components or through changes in the three-dimensional (3D) microtumor integrity [10-13]. In SCLC and certain neuroendocrine tumors, cancer progression correlates with increased modification of the neural cell adhesion molecule (NCAM; NCAM1; or CD56) by polysialic acid (polySia) [1]. PolySia-NCAM (alternatively referred to as PSA-NCAM; denotes an “embryonic” form of NCAM) presents a tumor-restricted [1] but heterogeneously expressed therapeutic target [1]. The relationships between polysialylation, proliferative capacity, and adhesion potential appear complex demanding single-cell population profiling. Our aim was to apply flow cytometry to assess the dynamic nature of polySia-NCAM expression in live SCLC cells during spontaneous transition to adherence. We have used a classical SCLC cell line model, NCI-H69, to determine the extent of shifts in polysialylation and their impact on cell proliferation both in vitro and in vivo.

PolySia is a linear α-2,8-linked polymer of up to 200 residues of N-acetylneuraminic acid (sialic acid, Neu5Ac) assembled on the IgV domain of NCAM by the Golgi-associated polysialyltransferases, ST8SiaII (STX), and ST8SiaIV (PST) [1]. The embryonic form polySia-NCAM, virtually nonexistent in normal cells by adulthood, is conventionally detected using antibodies that recognize the polySia epitope in context with NCAM. Polysialylation is thought to increase the amount of intercellular space, providing intermembrane repulsion [1], and facilitating neuronal plasticity [1]. Polysialylation may influence adherence properties [8, 20] or cell migration via ECM adhesion, pointing to roles in cell detachment and metastatic migration [1]. PolySia can undergo processive endosialidase cleavage by bacteriophage borne glycosyl hydrolases [review: [1]]. Certain endosialidase mutants devoid of polySia cleaving capacity retain high-affinity substrate binding. When fused with eGFP these “inactivated” enzymes can provide convenient tools for the detection of polySia on live cells [22, 23]. The antibody mimetic EndoN-GFP probe [1] rapidly binds to polySia molecules. We rationalized that in combination with the nontoxic far-red fluorescent viability dye DRAQ7 [24-27] excluded from intact cells, the antibody mimetic would allow a real-time approach for assessment of polySia expression in complex cell populations undergoing adherence transition.

Classical NCI-H69 SCLC cell cultures were successively enriched for subpopulations with APs and then assessed for their heterogeneity in NCAM polysialylation, using live and fixed cell flow cytometry, and for ECM selectivity using multiple substrate arrays (MSAs). Adherent forms were assessed for changes to in vivo growth as subcutaneous xenografts. We report that striking changes in polySia expression accompanies the increasing selection for substrate adherence with evidence that such states are affected by microcommunity composition. Homeostasis is in part maintained by differential proliferation rates. Enhanced rapid capture of adherent and nonadherent polySia-positive cells on different ECM components was not significantly affected by polySia removal suggesting that polySia expression in SCLC presents a dynamic target independent of adherence potential. Breaking the linkage between polySia expression and spontaneously acquired adherence was further supported by profiling polysialylation in a panel of SCLC cell lines.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. Literature Cited
  8. Supporting Information

Cell Culture

Panel cell lines and cell culture have been described previously. Cells were resuspended using nonenzymatic cell dissociation solution (CDS; Sigma, St Louis, MO) [28-32]. SCLC cell lines were originally established from: pleural effusions, NCI-H69, COR-L51, and COR-L88; lymph nodes COR-L47 and COR-L279; primary tumor SHP-77 (CRL-2195). The polySia-negative large-cell lung cancer line COR-L23 was established from a pleural effusion. COR-designated cell lines were obtained from the originating laboratory (Dr P Twentyman; formerly MRC Clinical Oncology and Radiotherapeutics Unit, Cambridge, UK); authenticated SHP-77 (CRL-2195) and NCI-H69 were obtained from the American Type Culture Collection.

Variant Enrichment and Culture

Tissue culture (TC) plastic adherent phenotype (AP) populations of COR-L88, COR-L279, SHP-77, and NCI-H69, were isolated from the parental suspension phenotype (SP) parental populations and cultured separately biweekly before analysis. The enrichment procedure for NCI-H69 generated a suspension form (i.e., NCI-H69 SP) and two AP sublines: an “early transition state” subline NCI-H69 AP3 (nominally passage 3 but <10), and by continuing selection for substrate adherence a stable, fully adherent subline NCI-H69 AP78 (nominally passage 78; representing >150 population doublings). To quantify cell attachment rates, cells were plated in triplicate at 4 × 106 cells per T25 TC plastic flask and cultured and counted. TC cultures were also transferred to hydrophobic (HP) bacteriological quality Petri dishes (Corning, Fisher Scientific, Loughborough, UK) being re-fed without disturbing the aggregates (initiating cultures of 5 × 105 cells/10 mL/9-cm dish).

Immunofluorescence and Q-Tracker® 705 Flow Cytometry

General flow cytometric and confocal immunofluorescence procedures have been described previously [1]. Primary antibodies: polySia-NCAM rat IgM (12F8; BD Bioscience Pharmingen, San Diego, CA); polySia mouse IgM (2-2B; AbCys, Paris, France); NCAM mouse IgG1 (B159, BD Bioscience Pharmingen, San Diego, CA). Immunostaining used anti-mouse Alexa-488 and anti-rat Alexa 488 secondary antibodies (Invitrogen, Carlsbad, CA). Q-tracker® 705 Cell Labeling Kit (Invitrogen, Carlsbad, CA) was used to load (1 h) near-infrared-fluorescent Qdot® 705 nanocrystals into the cytoplasm of live NCI-H69 AP78 cells and analyzed as described previously [1]. To determine the cell population distribution of Q-dot 705 fluorescence intensity a FACScan flow cytometer was used (Becton Dickinson, Cowley, UK) which was equipped with an air-cooled argon ion laser (with 488-nm output only). CELLQuest software (Becton Dickinson Immunocytometry Systems) was used for signal acquisition [1]. The supporting information MiFlowCyt document details the flow cytometer instrument set-up and configuration for a FACS Vantage flow cytometer system (Becton Dickinson, Cowley, UK) equipped with a Coherent Enterprise II argon ion laser (488-nm output alone selected) for immunofluorescence.

Paraffin-Embedded Sections of Cell Aggregates for Fluorescence Microscopy

Paraffin embedding, sectioning, and dewaxing were performed in collaboration with Dr J.M. Morgan (University Hospital of Wales). Cells were grown on (HP) surfaces for 7 days, then centrifuged, washed with cold PBS once, and fixed overnight in 10% formalin in isotonic PBS at pH 7.0–7.2. After fixation at room temperature for 12–16 h, the cells were allowed to settle out from suspension and the cell pellet was transferred to a 1.5-mL microcentrifuge tube. The pellet was washed twice in PBS, before adding 50 μL of 4% agar at 60°C. When set, the agar containing the cells was then processed for paraffin embedding by sequential dehydration, of 1 h in each, in ethanol series, 10–100%, followed by immersion in xylene, four washes of 1 h each. The agar/cell pellet was then processed to a paraffin block and 4-μm sections cut. Sections were dewaxed, washed, and processed for immunofluorescence staining with an anti-polySia antibody (i.e., anti-PSA primary antibody clone 2-2B) and an anti-mouse Alexa-488 secondary, and for nuclear DNA detection with DRAQ5 (20 μM) before fluorescence microscopy.

EndoN-GFP Detection of Cell Surface PolySia by Direct Flow Cytometry

The polySia antibody mimetic EndoN-GFP [1] probe is a noncatalytic bacteriophage-derived endosialidase fused to EGFP (1.37 mg/mL; provided by Prof. Jukka Finne; University of Helsinki, Finland) and was added directly to cells in full medium with 10 mM HEPES (5 × 106 cells/mL; 10 μg/mL) for 20 min at room temperature in the dark. Samples were made up to 5 × 105 cells/mL before the addition of the dye DRAQ7 at 3 μM for the determination of cell integrity by dye exclusion (Biostatus, Shepshed, UK) and direct analysis by flow cytometry for 488-nm excitation as previously described [33, 35] for cell populations gated for normal light scatter characteristics. Accompanying MiFlowCyt document details the flow cytometer instrument set-up and configuration for a FACS Vantage flow cytometer system (Becton Dickinson, Cowley, UK) equipped with a Coherent Enterprise II argon ion laser (488-nm output alone selected).

Neuraminidase Digestion of PolySia and Multiple Substrate Arrays

Endoneuraminidase (endo-N; AbCys S.A., Paris, France) is a nontoxic bacteriophage enzyme that specifically cleaves polySia and is active in culture medium [1]. Live cells (2 × 106) were seeded in T25 flasks for 24 h before addition of 0.7 units/5 mL medium of endo-N or heat-inactivated (80°C for 15 min) endo-N and overnight incubation (12–15 h). MSAs were applied as recommended [1]. Arrays comprised: serum albumin bovine, fibronectin (source: human plasma), fibronectin cellular (source: human fibroblasts), vitronectin, heparan sulfate proteoglycan, laminin (source: human placenta), laminin (source: basement membrane of Engelbreth-Holm-Swarm murine sarcoma), and Type I–VI collagens. Briefly, MSA slides were mounted in a ProPlate multiarray slide module (Grace BIO-LABS, Bend, Oregon), prepared in serum-free medium and tested for rapid cell capture (2 h) as determined by microscopy.

SDS-PAGE/Immunoblotting

Cell lysates were analyzed on 7.5 or 12% SDS-PAGE (Bio-Rad, Hemel Hempstead, UK), transfer onto Hybond-P PVDF membrane (GE Healthcare, Little Chalfont, UK) and antibody probing. Primary antibodies were polySia-NCAM [rat IgM;12F8; BD Bioscience Pharmingen (San Diego, CA)]; polySia [mouse IgM; 2-2B; AbCys (Paris, France)]; NCAM [mouse IgG1; B159; BD Bioscience Pharmingen (San Diego, CA)], and GAPDH [mouse IgG2b; Abcam (Cambridge, UK)]. Secondary HRP-conjugated were rabbit anti-mouse (0.1 μg/mL; DakoCytomation, Ely, UK) or goat anti-rat (0.33 μg/mL; Jackson ImmunoResearch, West Baltimore Pike, PA). Proteins detected using an Advance Detection Kit (GE Healthcare, Little Chalfont, UK) and image capture.

In vitro Invasion Assay

Basement membrane matrix invasiveness potential of the sublines was tested using a 48-h in vitro Cell Invasion Assay (Calbiochem, San Diego, CA) according to manufacturer's instructions (upper chamber loads of 3 × 105 cells; lower chambers loaded with medium supplemented with 10% FCS).

In vivo Tumourigenicity

Male Balb/c immunodeficient nude mice (Harlan, Loughborough, UK) aged 8–12 weeks were used. Mice received Harlan 2018 diet (Harlan) and water ad libitum according to guidelines [1]. Tumor cells in cell culture medium were adjusted to a concentration of 1 × 108 cells/mL and 100 μL was injected subcutaneously in left and right flanks of each mouse, with five mice inoculated/cell type. Palpable tumors were assessed as previously described [1]. Tumor take was determined by the time to establishment of a tumor of minimal size of 60 mm3. Relative proliferative rate of each tumor was determined by an exponential fit [y = a(ebx)] of the data tumor volume (y) versus time (x), data extracting the exponent b and applying single factor ANOVA to the three sample groups of b values to test whether the means of the groups are all equal (P < 0.05).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. Literature Cited
  8. Supporting Information

Immunodetection of Changes in NCI-H69 NCAM Polysialylation During Enrichment for Adherence

Figure 1A shows the appearance of the classical suspension growth form of NCI-H69 (SP) and sublines. An early phase of enrichment for the adherence transition state, over ∼10–30 population doublings (on TC plastic; TC, generated the adherent “subline” NCI-H69 AP3 (AP3). Longer-term passage over >150 population doublings of the AP3 subline, with continued selection for adherence, generated the highly adherent subline NCI-H69 AP78 (AP78). Figure 1B confirms the differences between the sublines for a rapid attachment phenotype on a TC substrate, with NCI-H69 AP78 (∼70% cells) > AP3 (∼30% cells) > SP (<5% cells) showing the presence of adherent fractions at <6 h.

image

Figure 1. Changes in NCAM polysialylation during enrichment for adherence. (A) Transition adherence model for NCI-H69 cells. Brightfield images of NCI-H69 (on TC plastic) following selection for growth as aggregates in suspension (NCI-H69 SP), enrichment for early transition adherence (NCI-H69 AP3), or continued selection for proliferative capacity and adherence (NCI-H69 AP78). Scale bar shown for all images = 50 μm. (B) Typical time-dependent adherence of sublines to TC plastic. (C) Fluorescence microscopy of NCAM (antibody B159) and polySia-NCAM (antibody 12F8) expression. Cell surface coexpression arrowed, with minor subpopulations of polySia-NCAM-positive and -negative cells in NCI-H69 AP3 and AP78, respectively. Scale bar shown for all images = 50 μm. (D) Flow cytometric analysis of NCAM (antibody B159) and polySia-NCAM (antibody 2-2B) expression confirms the overlap in NCAM expression but the reacquisition of polySia-NCAM positivity in NCI-H69 AP78.

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Preliminary in situ fixed cell immunofluorescence (Fig. 1C) indicated consistent NCAM expression located at the cell surface including cell–cell contacts for all three forms. As expected SP cultures were polySia positive but showed a low background of dim cells essentially polySia-NCAM negative. AP3 reproducibly yielded a polySia-NCAM-negative population with a low-frequency background of infrequent adherent but highly polySia-NCAM-positive cells (Fig. 1C arrowed). This background is the likely origin of the AP78 form that was found to extensively express polySia-NCAM. When expressed, polySia-NCAM colocated with NCAM at cell–cell boundaries. Flow cytometry of fixed cells confirmed identical NCAM expression for the three cell lines (Fig. 1D; typical data shown; n > 5) and the patterns of polysialylation with AP3 reproducibly presenting as a polySia-NCAM-negative population (n > 5), while A78 showed extensive polySia-NCAM high expression.

Rapid Adhesion to ECM Substrates is Independent of Polysialylation

Using immunoblotting we next confirmed (Fig. 2A) that: (i) the general polysialylation levels for the three cell lines could be reiterated, (ii) the appearance of a polydisperse band with high molecular weight for NCAM extracted from SP and AP78 cells, but not AP3 cultures, indicated protein modification, and (iii) enzymatic cleavage of polySia from cell surface NCAM (using an active endoneuraminidase) resulted in a loss of the polydisperse bands revealing similar levels of NCAM expression for the three cell lines. Using the ability to remove polySia-NCAM from live cells using active endoneuraminidase, we determined if rapid capture was dependent upon polysialylation for different ECM substrates (Fig. 2B). The results show that adherence on laminin was preferred by all forms irrespective of enzymatic removal of polySia. The highly adherent AP78 cells showed a lower level of competence for adherence for other ECM components (vitronectin > fibronectin > HSPG). ECM adherence ranked in the order SP < AP3 < AP78, irrespective of endo-N treatment, showing that polysialylation does not limit rapid capture on ECMs.

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Figure 2. Adherence of NCI-H69 sublines to extracellular matrix (ECM) components. (A) Immunoblots confirming digestion of polySia on live cells by incubation with an active endoneuraminidase (endo-N); [NCAM (antibody B159); polySia-NCAM (antibody 2-2B); GAPDH loading control]. (B) Relative MSA adhesion profiles (n = cell number) of endo-N or heat-inactivated endo-N (control)-treated cells.

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EndoN-GFP Detection of Live Cell Polysialylation During Adherence Transition

Using NCI-H69 cells, we established the approach for profiling live cell binding of the EndoN-GFP probe (Figs. 3A–3I). Figures 3A and 3B show the region discrimination for EndoN-GFP binding to cells identified as DRAQ7− or DRAQ7+. Loss of plasma membrane integrity is associated both with DRAQ7+ staining and loss of EndoN-GFP binding indicating the need for a viability indicator when profiling loss of EndoN-GFP binding. Compared to the SP control population, the relative frequency of intact AP3 cells identified as EndoN-GFP positive was greatly reduced while AP78 cells show an enhanced frequency, Fig. 3C). These results are consistent with the findings using the polySia-NCAM antibody.

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Figure 3. EndoN-GFP profiling of NCAM polysialylation using viability monitoring and doublet discrimination. (A) Typical detection of the background of nonviable DRAQ7+ cells in NCI-H69 SP cultures. (B) Nonviable cells are EndoN-GFP−. (C) Proportion of EndoN-GFP+ viable cells in NCI-H69 populations changes upon enrichment for adherent forms. Both AP3 cells and suspension forms arising from adherent AP3 cultures (i.e., AP3/susp) demonstrate negligible EndoN-GFP fluorescence. (D) Typical example identification of single cells in NCI-H69 AP78 cell populations using pulse analysis for nuclear DNA DRAQ5 fluorescence. (E) Fractions of EndoN-GFP-positive cells presenting as singlet or doublet forms in NCI-H69 sublines. (FI) Distributions for EndoN-GFP binding to NCI-H69 subline populations.

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We observed that adherent AP3 cultures (see Fig. 1A) presented rounded forms, indistinguishable from the SP forms, which were typically attached to the surface of the adherent “islands” of cells rather than the substrate itself. These forms presented an opportunity to test if cell–cell contact rather than substrate adherence per se might influence polySia expression. These “AP3/susp” forms could be readily detached mechanically from adherent islands for direct analysis to determine whether they had reverted to SP-like polysialylation or showed AP3-like loss of polysialylation. These AP3/susp cells were found to be EndoN-GFP negative (Fig. 3C).

To determine whether tight cell–cell adherence affected polySia expression, cultures were gently resuspended using nonenzymatic cell dissociation solution such that a background remained of cell clusters comprising two to three cells. We used a conventional “doublet” discrimination method (pulse area vs. pulse width for DRAQ5 staining of cellular DNA of all cells) to identify clusters with extended pulse width characteristics (Fig. 3D). Doublet frequencies were similar in SP and AP78 cultures (∼30%) but reduced in AP3 and AP3/susp (∼10%) (Fig. 3E). AP78 samples showed an increase in the frequency of EndoN-GFP+ singlets compared with SP samples (Fig. 3E). For AP3 and AP3/Susp samples, the majority of events were found to be EndoN-GFP− singlets. Frequency distributions (Figs. 3F–3I) for EndoN-GFP fluorescence intensities consistently showed the predictable increase in signal for doublets versus singlets, suggesting that tight cell–cell adherence does not compromise polySia expression in either SP or AP78 populations.

Figures 3F–3I demonstrate that the patterns for EndoN-GFP binding for NCI-H69 SP, AP3, and AP78 cells reiterate the patterns revealed using immunodetection. However, typical contour maps for EndoN-GFP binding versus cell viability (Fig. 4) permit a simple approach of quadrant analysis to distinguish: the subfraction of EndoN-GFP−/DRAQ7− intact cells resident in SP cultures (∼9%) within a greater background of irrelevant EndoN-GFP−/DRAQ7+ nonviable cells (∼16%); the >97% (AP3) and >88% (AP78) purity for loss or retention of EndoN-GFP positivity, respectively. The EndoN-GFP+ AP78 cell population identified. The results shown in Figure 1I indicate that AP78 retains a minor subfraction of low (dim) EndoN-GFP singlets overlapping the AP3 profiles (Fig. 3G). DRAQ7 analysis (Fig. 4) shows that indeed such cells exist (∼4%) and appear viable, suggesting that even after <150 population doublings under adherence selection heterogeneity remains.

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Figure 4. Representative flow cytometric contour plots of EndoN-GFP (polySia expression) versus DRAQ7 (viability) staining of live NCI-H69 sublines cultured on tissue culture plastic (gated for normal light scatter).

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Proliferation and Polysialylation in 2D versus 3D In Vitro Culture

The findings suggest that NCI-H69 cells have a propensity to undergo adherence transition with concomitant loss of polysialylation while establishing a dynamic balance in suspension populations in which such underpolysialylated cells do not overwhelm the culture. We suggest that the balance could reflect ongoing transition to adherence with a degree of stability for that phenotype but with concomitant growth suppression. Proliferative restraint may reflect growth potential in two-dimensional (2D) versus 3D culture compounded by the effects of reduced polysialylation on NCAM signaling [1] or altered efficiency of autocrine stimulation.

To test the impact of culture state on polysialylation patterns and proliferative capacity, NCI-H69 cells were cultured either on TC or on (HP) surfaces, the latter preventing substrate attachment and enforcing the growth of all cells as classical aggregates. Immunoblotting confirmed that overall polysialylation and NCAM patterns of the sublines were unaffected by the growth conditions (Fig. 5A). Growth rates ranked as AP78 > SP > AP3 (Fig. 5B) irrespective of 2D versus 3D culture. We conclude that initial enrichment for adherence yielded cells with reduced proliferative potential and low polysialylation. Moreover, both polysialylation changes and growth rate differences found in these enriched populations are not artifacts of the method of cell culture.

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Figure 5. PolySia-NCAM changes during adherence transitions. (A) Immunoblots showing that NCAM expression (NCAM antibody B159) and polysialylation patterns [polySia-NCAM (antibody 2-2B)] are unaffected by enforced growth of adherent forms on HP surfaces as 3D clusters. (B) Substrate-independent growth on HP surfaces does not affect proliferation of NCI-H69 sublines. (C) Fluorescence microscopy images of representative sections of fixed, paraffin-embedded NCI-H69 AP78 spheroids showing polySia-NCAM expression (polySia-NCAM antibody 2-2B) and cell nuclei staining (DRAQ5). Scale bar shown for all images = 100 μm. (D) Cumulative events versus fluorescence plots of NCI-H69 AP78 cells loaded with Qdot® 705 nanocrystals then grown on TC or HP substrates.

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We noted that the highly AP of AP78 cells resulted in the rapid aggregation and elaboration of compact multicellular clusters (spheroids) when grown on HP surfaces indicating that enhanced substrate adherence translated into extensive cell–cell contacts. Even within such clusters polySia-NCAM expression was maintained throughout the cluster layers and only lost by moribund detached cells in the central regions (Fig. 5C) consistent with the loss of EndoN-GFP binding in DRAQ7+ cells (Fig. 4). These findings are also complementary to the singlet-doublet study above in suggesting that polySia expression per se does not preclude efficient cell–cell contact. To confirm that proliferation did not “stall” in a subset of AP78 cells as a consequence of such growth in 3D, patterns of cell division were profiled by the proliferative dilution of Qdot® 705 nanocrystals (QDot705) through a lineage [34, 41]. QDot705-loaded AP78 cells were plated onto TC or HP surfaces (Fig. 5D) generating 2D and 3D cultures, respectively. The attenuation of the QDot705 signal distribution with time was essentially the same for TC or HP AP78 cultures cells after 1–5 days of culture. Thus, NCI-H69 AP78 cultures maintain both efficient polysialylation and initial proliferative capacity even during the formation of tight cell–cell contacts and the establishment of a 3D spheroid structure.

In Vivo Proliferation of NCI-H69 Xenografts

Using a conventional in vitro invasion assay (Fig. 6A), we determined that the SP, AP3, and AP78 forms all showed low invasion potential. This was in keeping with a preliminary assessment of subcutaneous NCI-H69 xenografts in Balb/c immunodeficient nude mice showing no evidence of metastasis even for tumor volumes in excess of 500 mm3. Using this xenograft model, we assessed the ability of the three phenotypes to establish and proliferate in vivo (Fig. 6B). The time taken to establish a tumor (in this case defined at >60 mm3 tumor volume; 10 xenografts assayed per subline) was measured over a 40-day period, together with the changes in tumor volume. The results (Fig. 6B) show that the mean time to tumor take ranked AP3 (26.6 days) > SP (19.6 days) > AP78 (15.5 days) [P = 0.187; single factor ANOVA between groups; F = 1.843]. Conversely, average proliferation rate (given by the exponent b extracted from exponential fits of tumor volume versus time; see Materials and Methods section) ranked AP3 (0.0483) < SP (0.0653) < AP78 (0.0870) [P = 0.0245; single-factor ANOVA between groups; F = 4.584). Thus, even within the restrictions of assessing in vivo growth potential, the sublines reiterated their rankings for in vitro establishment and growth (Fig. 6C) when tracked as xenografts for up to 40 days. Figure 6B indicates that the average proliferation rate of AP78 xenografts is significantly greater than that of AP3 xenografts (P < 0.05; Student's t-test). The results suggest that if adherence transitions occur in vivo and are accompanied by loss of polysialylation, they may be persistent but occult due a reduced proliferative capacity.

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Figure 6. Invasion and in vitro growth of NCI-H69 sublines. (A) Low and similar in vitro invasion properties of NCI-H69 SP, AP3, and AP78 cells (control = 1 × 105 cells in the invasion chamber). (B) In vivo growth (columns) and time to xenograft establishment (line) for NCI-H59 sublines grown on TC plastic before inoculation into mouse flanks. (C) Typical hematoxylin and eosin-stained sections of tissue surrounding the injection site taken 72-h postinoculation of NCI-H69 AP78 cells. AP78 deposits of viable cells are clearly seen with evidence of mitotic activity (arrows) seen at a higher magnification. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com.]

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Dynamic Changes in Polysialylation Within NCI-H69 Microcommunities

The findings suggest that polysialylation is not modified by the realization of substrate adherence per se. However, the above studies necessarily use enrichment methods that separate out phenotypes from mixed populations that may otherwise undergo unknown homeostatic influences. We hypothesized that population composition could modify cellular polysialylation status. To test this directly, nominal 1:1 mixtures of SP and AP3 cells were cocultivated to construct a situation where the AP3 forms were approximately fivefold over-represented. To control for any influence of culture method, upon admixture cultures were established either on a TC substrate to allow cells to realize any adherent potential or on a HP substrate to enforce a mixed 3D growth in aggregates. Based on proliferation capacities alone, the prediction would be for a diminution in the frequency of underpolysialylated forms (i.e., AP3) as cocultivation progressed in favor of the polysialylated forms (i.e., SP) during a typical cell-culture passage period (6 days). Furthermore, such changes in frequency should conform to a simple arithmetic principle if the component populations acted independently of each other.

EndoN-GFP/viability contour plots (Figs. 7A and 7B; exemplar results for growth on TC substrate) confirmed the stability of the EndoN-GFP profiles for control AP3 and SP monocultures and the ability of DRAQ7 positivity to track changes in polySia-negative moribund cells (Fig. 7A, lower right quadrants). Contrary to prediction, following admixture there was a decrease in the frequency of viable polySia-NCAM-positive cells (38–16%) not attributable to cell death. K–S statistical analysis (Fig. 7C) was used to compare the EndoN-GFP distribution for coculture (experimental) with a simple arithmetic “combination” of EndoN-GFP distributions derived from the parallel SP and AP3 monocultures without any adjustment for relative proliferation rate. The results clearly show an early increase in frequency of EndoN-GFP dim cells (e.g., increased D values 0.43 at low channel number 21; P < 0.01) separable from the AP3 expression range. By Day 6, a reduction in the frequency of events in the high-level expression SP range was also apparent. The results support the hypothesis that polySia expression is influenced by SCLC microcommunity composition independent of substrate adherence.

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Figure 7. Polysialylation changes in reconstructed microcommunities containing increased levels of adherent forms. (A) Representative flow cytometric contour plots of EndoN-GFP versus DRAQ7 staining of live NCI-H69 (gated for normal light scatter) during enforced substrate-independent growth (HP surface) of NCI-H69 SP, AP3, or ∼1:1 combination, showing a decrease in the frequency of viable polySia-NCAM-positive cells (38–16%) following admixture. (B) Corresponding analysis of polySia expression shows acquisition of low levels of polysialylation by NCI-H69 AP3 cells. (C) Corresponding K–S statistical analysis comparing the distributions of polySia expression in the experimental mixed cultures (∼55% AP3 at t = 0) versus the calculated combinations (∼65% AP3 at t = 0; derived from the arithmetic combination of the distributions of the isolated NCI-H69 SP and AP3 populations). Channel number of D (Kolmogorov–Smirnov statistic) at which the greatest difference between the two curves occurs; P < 0.001); D/s(n), an index of similarity for the two curves [if D/s(n) = 0, the curves are identical]. The acquisition of low-level expression and reduced frequency of high-level expression was apparent irrespective of the attachment substrate.

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NCAM Polysialylation Patterns of SCLC Cell Forms

The results suggest that if adherence per se is functionally independent of polysialylation level then it should be possible to identify early adherence transitions associated with increased polysialylation in a SCLC panel. We examined several SCLC cell lines that grew typically as nonadherent loose aggregates (i.e., suspension phenotype; SP) [28, 42]. COR-L88, COR-L279, and SHP-77 consistently generated a minority of cells with an AP for TC plastic. Separation of SP and AP forms resulted in retention of antibody-detectable NCAM expression as determined by flow cytometry (Fig. 8A; compared with the NCAM-negative adherent large-cell lung cancer control cell line COR-L23). Immunoblotting revealed the appearance of polydisperse bands with high molecular weight for NCAM in SHP-77 AP but very low levels of NCAM polysialylation apparent in the separated SHP-77 SP. COR-L279 SP cells showed moderate levels of NCAM polysialylation, similar to that of the NCI-H69 SP control, but a further enhancement of polysialylation in adherent forms. Using DRAQ7 negativity to exclude moribund cells (Fig. 8C), live COR-L279 and SHP-77 cell populations were assayed for EndoN-GFP fluorescence binding revealing striking increases in mean polysialylation levels (P < 0.05) for cells showing transition to adherence, completely consistent with the immunoblot patterns.

image

Figure 8. NCAM polysialylation patterns of adherent forms of SCLC cell lines. (A) Flow cytometric analysis of mean NCAM expression (± SD; n = 3) for a SCLC cell line panel including cultures initially enriched for either adherent (AP) or nonadherent growth (suspension phenotype; SP). The large cell-lung cancer cell-line control COR-L23 provides a non-NCAM expressing control. COR cell lines L47 and L51B grew exclusively as aggregates in suspension. (B) Immunoblot analysis of NCAM (antibody B159) and polySia-NCAM (antibody 2-2B) protein expression in the SCLC cell line panel. (C) Viable cell (DRAQ7 negative) population expression of polySia-NCAM (range; antibody mimic EndoN-GFP). (D) Upper panels: transmission images (field 300-μm wide) of typical appearance of SHP-77 and COR-L279 cultures initially enriched for AP or SP forms (scale bar shown for all images = 50 μm). Middle and lower panels: typical flow cytometric contour plots of EndoN-GFP versus DRAQ7 staining for sublines enriched for SP or AP forms.

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Figure 8D shows the morphological appearance of the transition forms and the linked EndoN-GFP/viability contour maps. The results clearly show that acquisition of adherence results in changes in EndoN-GFP-positive cells from 13 to 62% and 83 to 92% for SHP-77 and COR-L279, respectively. The gain in polySia expression for the adherent form was striking. SHP-77 is known to be an unusual undifferentiated large cell variant of small-cell lung carcinoma that typically grows as floating clusters that consistently generate loosely adherent cells as observed in the current study. Only floating cells are typically transferred but even after more than 200 passages SHP-77 cultures retain these AP-generating properties described for the original cell line [32, 43]. The results indicate that such SHP-77 variants are enriched in polySia expressing cells. Overall, the findings clearly indicate that in concert with the NCI-H69 data, adherence transition per se is not predictive of the loss or gain of polysialylation.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. Literature Cited
  8. Supporting Information

Our cytometric approach attempted to address the ability of SCLC cells to acquire and maintain new phenotypes within a parental microcommunity, with a focus on a potential therapeutic target—NCAM polysialylation. The approach was different to that described in a previous study of the characteristics of stable clonal SCLC cell lines with different levels of polySia expression [1] in which there was no imposition of selective pressure for adherence. We have brought a focus to bear upon the impact on polySia expression of enrichment for an emergent AP. The findings reveal that SCLC can generate variant subpopulations with high or low adhesion potential that can independently present as polySia-positive and -negative states.

It is appreciated that a role for polySia in cell adhesion is supported both by the paradigm of neuronal plasticity and by observations that for other cell types endoneuraminidase-mediated removal of polySia can increase cell–cell aggregation [1] and cell adhesion to laminin [1], cadherin, fibronectin, and collagen IV [45, 46]. In the case of SCLC, we found that polySia removal in parent and adherent NCI-H69 cells did not enhance rapid capture on a range of ECM substrates particularly fibronectin, vitronectin, and laminin. This breaking of a connection between SCLC cell-substrate adherence and polysialylation is consistent with an earlier report showing that clonal SCLC sublines with different levels of stable polySia expression did not differ in their attachment to collagen type IV, laminin, heparan sulfate, and poly-L-lysine [1]. Using the approach of physically removing polySia by enzymatic cleavage tests the functional dependency of a characteristic on polysialylation but does not reiterate the extent to which the dynamic acquisition of adherent properties is favored by reduction in polySia expression. Importantly, the SCLC cell-line panel showed that acquisition of adherence was not predictive of reduced polysialylation although NCAM expression was consistently maintained. The breaking of any obligatory linkage between ECM adherence and polySia expression suggests that the latter will not primarily limit ECM-mediated drug resistance in SCLC [1].

A previous report indicated that high polySia expression correlated with reduced cell–cell adherence [1], a feature clearly not shared by the exemplar of NCI-H69 AP78 in the current study of the reduced doublet frequency in polySia-negative NCI H69 AP3 cells. Rather, our study indicates that selection for adherence competence can impose a dominating influence over any reduction in cell–cell adherence attributable to the polyanionic surface charge donated by polysialylation. Indeed, SCLC cells are already primed for adherence by constitutively phosphorylated focal adhesion kinase [1]. Enhancing cell–cell contact for adherent SCLC cells by enforcing growth of polySia-positive cells in 3D tight clusters, versus 2D substrate attachment, did not impact on relative levels of polysialylation or proliferation rate. Further, we found that in vivo tumor growth patterns for isolated NCI-H69 subpopulations reflected relative in vitro proliferation in either 2D or 3D culture formats and that proliferation ranked positively with polySia expression. This is consistent with the observations of polySia control of tumor growth through the masking of heterophilic NCAM signals [1].

Skewing population composition for polySia-positive and -negative subpopulations in coculture experiments resulted in unexpected polySia expression profiles, suggesting that cells do not act independently and can be influenced by neighboring cells to modify polysialylation. This influence appears to be independent of any adherence to a substrate. Intrinsic differences between subpopulation proliferative rates and differential cell death tracked using DRAQ7 could not account for the shift in polySia profiles presented in coculture. The results are consistent with an actual modulation of polySia expression in all cells. This could reflect neuroendocrine homeostatic influences acting between polySia-negative and -positive subpopulations within the microenvironment of coculture. In ancillary studies (data not shown), we have used global gene expression analysis to search for changes linked with adherence transition. This initial screen highlighted significant downregulation of gastrin releasing peptide (GRP; SP/AP3 = 84.7) and neurotensin (NTS; SP/AP3 = 18.1) genes in AP3. This could explain the reduced proliferation rate of AP3 cells through decreased autocrine stimulation when enriched from parental populations [1]. We suggest that a basis for further study would be to examine the extent to which neuropeptide hormones [49, 50] and receptor expression can direct changes in polySia expression in microcommunities.

A dynamic relationship between polysialylation status and adherence allows for the generation of multiple phenotypes in SCLC. In the absence of stable genetic variation, it is reasonable to attribute the findings of different subpopulation states in SCLC to the operation of complex networks of gene expression [51, 52]. Studies on gene regulatory networks have revealed “switch-like behavior” or “bistability,” for the toggling of a gene between two steady states and resulting in alternative cell behaviors or fates [1]. A perspective for the current findings is that dynamic changes in polySia-NCAM expression and substrate adherence in SCLC reflect bistability for both phenotypes. Shifts in polysialylation state could act to favor switching of a network to an adherent state without functional interdependence. Such alternative states are only apparent on cytometric population profiling, the temporal frequency of the different states being under the selective influence of polysialylation favoring proliferation. Here, we have validated the potential of a live cell probe for polysialylation in combination with a nontoxic viability sensor to assess switches in state in SCLC microcommunities with the potential to track downstream behavior.

Shifts in polySia expression constitute nongenetic phenotypic plasticity. These shifts could be critical for proliferation and progression at discrete points in time during spread while they could remain occult if only examined downstream following exit from originating microniches. It has been proposed that such nongenetic phenotypic plasticity may even accelerate evolutionary rate under selective pressure for proliferation rate, leading to the appearance of cells with genotypic changes [1]—a cellular motif for SCLC. We note that in nonlung cancer cell models, polySia appears to have cell migration promoting influences that do not depend strictly upon the degree of adhesion to ECM components [55, 56]. Thus, a further complex dynamic balance could exist between migratory-competent polySia-expressing and migratory-incompetent polySia downregulated forms in SCLC microcommunities. The cytometric approach described here could be used to track both changes in cell viability and polySia expression during cell migration and in screening responses to antimetastatic agents or indeed polysialyltransferase inhibitors [1].

Acknowledgments

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. Literature Cited
  8. Supporting Information

The authors thank Patricia Cooper (ICT, Bradford) for support with the in vivo studies. EndoN-eGFP was a gift from Prof. Jukka Finne, and was synthesized by Dr Anne Jokilammi and Maria Pajunen (University of Helsinki). We thank Dr J.M. Morgan for his help with the paraffin embedding of spheroids. Authors PJS, RJE and LHP declare that they are nonexecutive directors of Biostatus Ltd, the commercial supplier of DRAQ5 and DRAQ7—general laboratory reagents used in the current study. Authors MW, SDS, and RAF declare no conflict of interest.

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  6. Acknowledgments
  7. Literature Cited
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. Literature Cited
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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