Sponge Hybridomas: Applications and Implications

Integrative and Comparative Biology Integrative and Comparative Biology, volume 53, number 3, pp. 524–530 doi:10.1093/icb/ict032

Society for Integrative and Comparative Biology

SYMPOSIUM

Sponge Hybridomas: Applications and Implications Shirley A. Pomponi,1,* Allison Jevitt,† Jignasa Patel‡ and M. Cristina Diaz* *Harbor Branch Oceanographic Institute, Florida Atlantic University, Fort Pierce, FL 34946, USA; †Florida State University, Tallahassee, FL 32306, USA; ‡Oceanographic Center, Nova Southeastern University, Dania Beach, FL 33004, USA From the symposium ‘‘Assembling the Poriferan Tree of Life’’ presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2013 at San Francisco, California. 1

E-mail: [email protected]

Synopsis Many sponge-derived natural products with applications to human health have been discovered over the past three decades. In vitro production has been proposed as one biological alternative to ensure adequate supply of marine natural products for preclinical and clinical development of drugs. Although primary cell cultures have been established for many marine phyla, no cell lines with an extended life span have been established for marine invertebrates. Hybridoma technology has been used for production of monoclonal antibodies for application to human health. We hypothesized that a sponge cell line could be formed by fusing sponge cells of one species with those of another, or by fusing sponge cells with rapidly dividing, marine-derived, non-sponge cells. Using standard methods for formation of hybridomas, with appropriate modifications for temperature and salinity, cells from individuals of the same sponge species, as well as cells from individuals of two different sponge species were successfully fused. Research in progress is focused on optimizing fusion to produce a cell line and to stimulate expression of natural products with therapeutic relevance. Experimental hybridomas may also be used as models to test hypotheses related to naturally occurring sponge chimeras and hybridomas.

Introduction Sponges constitute one of the most primitive and oldest living metazoan phyla and are one of the most diverse and abundant groups of benthic organisms. Marine sponges are a rich source of bioactive compounds with applications to human health; more than 7000 sponge-derived chemicals have been discovered over the past four decades (Blunt et al. 2009). Despite the biotechnological potential of sponge-derived bioactive compounds, only a few have been developed or are in clinical trials. A major obstacle is the lack of sufficient supply of the products for preclinical and clinical development. In vitro production is an attractive option for biological supply because it allows for control of culture conditions to increase both biomass and the concentrations of the product (Pomponi 1999, 2006). Although primary cell cultures have been established for many marine phyla, no cell lines with an extended life span have been established for marine invertebrates, including sponges (Rinkevich 2005).

We hypothesized that a secondary metaboliteproducing sponge cell line could be established by creating a sponge hybridoma, that is, by fusing non-dividing, somatic cells of a bioactive sponge with dividing cells of a non-bioactive sponge, or with rapidly dividing, marine-derived, non-sponge cells. Hybridomas were initially developed for the production of monoclonal antibodies by fusing non-dividing, antibody-producing mammalian cells with continuously dividing myeloma cells (Milstein 1999). Hybridomas differ from chimeras in one important aspect: cells are fused in hybridomas, but not in chimeras. A chimera is an organism with a mixture of cells from two or more genetically distinct individuals or species. Natural chimerism occurs in various marine benthic invertebrates, including sponges (Little 1966; Sara et al. 1966; Blanquer and Uriz 2011). This phenomenon has been used to address questions related to immune recognition (Johnston 1988; Gauthier and Degnan 2008), ecological fitness (Maldonado 1998), and estimates of

Advanced Access publication May 2, 2013 ß The Author 2013. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: [email protected].

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population genetic diversity (Blanquer and Uriz 2011). A hybrid is an organism resulting from the breeding of two different species, that is, the combination of germ cells, and is not known to occur in sponges. Hybridoma technology draws on both chimerism and hybridization: a cell is formed by the fusion of somatic cells of individuals of the same or different species.

Materials and methods Experimental animals The reef sponge CymbaxinellaP corrugata (Porifera, Demospongiae, Agelasida, Hymerhabdiidae) was collected by scuba diving from the reefs off Dania Beach, FL (with permission granted through a Florida Saltwater Fishing License issued to S.A.P.). This species produces a bioactive metabolite, stevensine, and has been used in our laboratory as a model for cell-culture research (Pomponi and Willoughby 1994, 2000; Pomponi et al. 1997). The sponges Haliclona sp. (Porifera, Demospongiae, Haplosclerida, Chalinidae) and Plakinastrella sp. (Porifera, Homoscleromorpha, Homosclerophorida, Plakinidae) were obtained from the recirculating seawater system maintained by Oceans, Reefs & Aquariums (ORAÕ ) in Fort Pierce, FL. They were selected because the sponges were easily accessible, and they were particularly amenable to laboratory culture and cell dissociation. Sponges were transported from the field or aquarium in filtered seawater (FSW) at 22–258C. Experimental protocols Dissociation and primary culture of sponge cells

Primary cell cultures were prepared immediately after collection. Each sponge was cut into fragments (3–5 mm3) and minced in 50 ml of sterile calciumfree and magnesium-free (CMF) seawater (Schippers et al. 2011) in a petri dish. The cells were filtered through a 40-mm nylon cell strainer into a 50-ml centrifuge tube. The filtrate was concentrated to 106–108 cells/ml by centrifugation at 1500 g for 10 min. Cell counts were determined microscopically using a hemocytometer. Primary cultures were maintained in T-flasks or multiwell dishes at room temperature (22–258C). For some of the fusion experiments, PercollÕ density-gradient centrifugation was used to obtain a less heterogeneous cell suspension and to remove bacteria, cell aggregates, and debris (Pomponi and Willoughby 1994, 2000; Pomponi et al. 1997).

Staining of cells

To identify cells from different individuals, dissociated and enriched cells from individuals of the same species (for intraspecific hybridization) or of two different species (for interspecific hybridization) were stained with either CellTrackerTM Green CMFDA, CellTrackerTM Red, or VybrantÕ DyeCycleTM Ruby (Molecular Probes) fluorescent dyes at working concentrations of 30, 100, and 5 mm, respectively. For staining with CellTrackerTM Green CMFDA and CellTrackerTM Red, dissociated and enriched cells (107 cells/ml) were centrifuged at 1200 g for 10 min and the supernatant discarded. One hundred microliters of stain was added to the pellet, the mixture lightly vortexed to mix the cells, and the stained cells incubated at 258C for 90 min. Cells were centrifuged again at 1200 g for 10 min and the supernatant discarded. One hundred microliters of CMF was added to the pellet, the mixture lightly vortexed and then incubated for 30 min at 258C. For VybrantÕ DyeCycleTM Ruby staining, 1 ml of the dye was added to dissociated and enriched cells (107 cells/ml in 0.5 ml of FSW), mixed in a 2-ml centrifuge tube, and lightly vortexed. Cells were incubated at 258C for 1 h. Fluorescence was verified using epifluorescence microscopy, with filters appropriate to the excitation and emission wavelengths for each stain (Table 1). Fusion of cells

Sponge hybridomas were established using techniques modified from mammalian hybridoma methods (Sanchez-Madrid and Springer 1986). Suspensions of dissociated and stained cells from two individuals (same or different species of sponge) were mixed in a 15-ml tube and centrifuged at 500 g for 6 min. After removing the supernatant, the tubes were gently tapped to loosen the pellet. To fuse the cells, 1 ml of 50% polyethylene glycol (PEG, Hybri-MaxTM, Sigma-AldrichÕ ) was added to the pellet drop-wise while stirring (Yang and Shen 2006). After adding the PEG, the mixture was stirred for an additional 2 min, then centrifuged at 500 g for 5 min. The supernatant was removed and cells were re-suspended in 100 ml of CMF. Each experiment was performed with three replicates. Experimental design Dissociated and enriched sponge cells were stained either green (CellTrackerTM Green CMFDA) or red (CellTrackerTM Red or VybrantÕ DyeCycleTM Ruby). The absorbance and emission maxima of CellTrackerTM Red did not adequately overlap with the excitation and absorbance spectra of the ‘‘red’’

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filter in the Agilent 2100 Bioanalyzer (Table 1), so VybrantÕ DyeCycleTM Ruby was selected as a replacement for CellTrackerTM Red in the quantitative analyses. Cells from individuals of the same or different species were mixed and fused using PEG. Unstained cells were used as controls for the stain, and mixtures of the stained cells without PEG were used as a control for hybridization. Fusion was qualitatively analyzed for all species-pairs using confocal epifluorescence microscopy. Quantitative analyses using flow cytometry (Agilent 2100 Bioanalyzer) were performed for both the intraspecific and interspecific Haliclona sp. and Plakinastrella sp. speciespairs. They were not performed for intraspecific and interspecific experiments with C.P corrugata because the flow cytometer was not available when we had primary cultures of C.P corrugata. Flow

Table 1 Absorbance/excitation and emission spectra of stains and Bioanalyzer filters Absorbance/ excitation (nm)

Emission (nm)

CellTrackerTM Green CMFDA

350–492a–550

480–517a–650

CellTrackerTM Red

450–577a–640

560–602a–750

Vybrant

Õ

DyeCycle

TM

Ruby

Bioanalyzer ‘‘blue’’ filter Bioanalyzer ‘‘red’’ filter a

Maximum wavelength (nm).

a

500–633 –700

625–670a–850

458–470a–482

510–525a–540

a

620–630 –696

674–680a–696

cytometry data are only presented for the interspecific Haliclona sp.—Plakinastrella sp. species-pairs. Each experiment was performed with three replicates; quantitative analyses of the cells were performed twice for each treatment and control.

Results Intraspecific fusion of cells from two individuals was successful for all three species (Fig. 1, qualitative data shown for Haliclona sp.) and interspecific fusion of cells from two individuals occurred between two species pairs: Haliclona sp.—C.P corrugata and Haliclona sp.—Plakinastrella sp. (Fig. 2, Table 2 for latter species-pair). Fusion between C.P corrugata and Plakinastrella sp. was not tested. Flow cytometry data were analyzed using two-dimensional dot plots of the intensity of fluorescence of each cell. To compare treatments and controls, the regions on the dot plots were separated by creating subsets, termed ‘‘gates,’’ represented by polygons on each graph in Fig. 2. Unstained sponge cells (controls) were gated below 100 relative intensity in ‘‘blue fluorescence’’ and between 100 and 103 relative intensity in ‘‘red fluorescence’’ (Fig. 2A and C). These gated regions of unstained cells were considered baselines, and the gates were inserted into the dot plots of the data for the stained cells (Fig. 2B and D). Any cells that fluoresced outside of the baselines were assumed to be fluorescing as a result of the cell

Fig. 1 Intraspecific fusion of two individuals of Haliclona sp. Cells were stained either red or green and observed using confocal epifluorescence microscopy. (A) Differential interference contrast; (B) red fluorescence; (C) green fluorescence; (D) overlapping confocal fluorescence image of fused cell (large arrow). Small arrow, unfused cell.

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Fig. 2 Two-dimensional dot plots of flow cytometry data showing the intensity of fluorescence of Haliclona sp. and Plakinastrella sp. cells. (A) Unstained cells of Haliclona sp.; (B) cells of Haliclona sp. stained with CellTrackerTM Green CMFDA; green gate, stained cells; black gate, unstained cells; (C) unstained cells of Plakinastrella sp.; gated regions indicate two subpopulations of autofluorescing cells; the upper region shows likely algal symbionts; the lower region shows likely autofluorescing sponge cells; (D) cells of Plakinastrella sp. stained with VybrantÕ DyeCycleTM Ruby; red gate, stained cells; black gate, unstained cells; (E) mixture of stained cells without PEG; green box, gated region of stained cells from B; red box, gated region of stained cells from D; arrow points to gated region containing cells fluorescing either red or blue, or both red and blue; F, mixture of stained cells with PEG; gates as indicated for E; arrow points to gated region containing cells that are fluorescing both red and blue.

Table 2 Quantitative data for the dot plots in Fig. 2

Treatment Region

Mixture of stained cells with no PEG (Fig. 2E)

Mixture of stained cells with PEG (Fig. 2F)

No. of Events

% Total

No. of Events

% Total

All events

3039

100.00

2906

100.00

All blue-fluorescing stained cells

13

0.40

13

0.40

All red-fluorescing stained cells

12

0.40

10

0.30

Stained cells fluorescing either red and green, or both red and green

12

0.40

10 (6 in ‘‘without PEG’’ gate and 4 in ‘‘with PEG’’ gate)

0.30 (0.20 in ‘‘without PEG’’ gate and 0.10 in ‘‘with PEG gate’’)

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tracker dyes (Fig. 2B, D, E, and F). Gates were then set up for the blue (Fig. 2B) and red (Fig. 2D) fluorescing cells, respectively, and then added to the plots for the cell mixtures without PEG (Fig. 2E) and with PEG (Fig. 2F). Unstained cells from both species of sponge fluoresced red (i.e., 4100 relative red fluorescence intensity) (Fig. 2A and C), suggesting that there were either autofluorescing microalgae or sponge organelles. We could not microscopically confirm the presence of autofluorescing sponge or algal cells in Haliclona sp. There appear to be two subpopulations of autofluorescent cells in Plakinastrella sp. (each gated in Fig. 2C), most likely, sponge cells and algal symbionts. Microscopic analysis confirmed the presence of autofluorescing algal symbionts, and based on the greater relative fluorescence of one of the subpopulations, we assume that the more strongly fluorescing cells (i.e., the cells within the purple fluorescent gate between 102 and 103 in Fig. 2C) are the algal symbionts. There is some overlap between gates for the cells that fluoresce red and those that fluoresce blue (Fig. 2E and F). There are a few cells in Fig. 2E (i.e., mixtures without PEG) that appear in the regions where cells could be fluorescing either both red and blue or only red or only blue; these cells are likely aggregates of two or more cells, each fluorescing only one color. The PEG graph (Fig. 2F) indicates a small number of cells fluorescing both red and blue, and although these cells are extremely rare (50.10% of the total cells counted; Table 2), corroborating microscopic analysis (e.g., Fig. 1) supports our conclusion that the cells are fused.

Discussion We have demonstrated that sponges are capable of forming both intraspecific and interspecific hybridomas. Development of a secondary metabolite-producing sponge hybridoma will require the hybridization of cells from a bioactive sponge with cells from an actively dividing cell line. Ideally, the cell line will need to have similar growth characteristics, particularly in terms of temperature and osmolarity. Use of mammalian cell lines is precluded because of the requirement for higher temperature and lower osmolarity of mammalian cell cultures. The use of insect cell lines, while similar in thermal requirements, is precluded because of the lower osmolarity of insect cell-cultures. We are testing the possibility of fusing sponge cells with algal cell lines, and, in particular, algae without cell walls. One advantage of a sponge-algal hybridoma is that the resulting fusant may be autotrophic,

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thereby reducing, or even eliminating, the need for development of optimized nutrient media. Some sponges contain intracellular algal symbionts (Hill et al. 2011), so there should not be any physiological impediments to fusing sponge and algal cells. We have preliminary data demonstrating that algal cells (Dunaliella tertiolecta) can be intraspecifically hybridized using the methodology described previously. Research continues in our laboratory to increase the efficiency of hybridization and to test the hypothesis that fusion of cells from two species of sponges that do not produce bioactive compounds will result in a hybridoma that produces therapeutically-relevant metabolites. Sponges are prolific producers of these secondary metabolites, many with antibiotic, immune-regulatory, and/or cell cycle-inhibitory properties (e.g., Blunt et al. 2009). Previous studies have demonstrated potential roles of sponge-derived secondary metabolites in deterring predation by fish (for review, see Pawlik 2011), in inhibiting larval fouling or microbial epibiosis (Kelly et al. 2005), and in providing chemical signals for reproduction (Bandaranayake et al. 1997). Their success in competing against other sessile, benthic invertebrates for substrate as well as the ability of some sponges to regulate immune responses (e.g., Bigger 1988; Mu¨ller et al. 1999) provide a basis for both naturally occurring chimerism and cell fusion. These phenomena have neither been systematically surveyed in nature nor have their ecological and biomedical potential been evaluated. The occurrence of both intraspecific and interspecific sponge chimeras in nature (Little 1966; Sara et al. 1966; Blanquer and Uriz 2011), the creation of experimental intraspecific (Maldonado 1998; Gauthier and Degnan 2008) and interspecific sponge consortia (Johnston 1988; this study), and the demonstration of both intraspecific and interspecific fusion of somatic cells (this study) lead us to speculate on the potential for occurrence of hybridomas in nature as well as more widespread occurrence of sponge chimeras. Regarding the latter, Blanquer and Uriz (2011) proposed the concept of ‘‘intraorganism genetic heterogeneity.’’ They suggested that this phenomenon could enhance survival of colonial sessile invertebrates with small populations and poor dispersal abilities, and that the increased size of chimeras could enhance their ecological fitness. They speculated that the accuracy of estimates of genetic diversity in sponge populations may be incorrect if chimerism is widespread. Gauthier and Degnan (2008) conducted an elegant study in which they tracked the development of

Sponge hybridomas

juveniles formed by the mixture of cells from larvae of two individuals of Amphimedon queenslandica. They demonstrated that two incompatible genotypes could coexist in chimeric post-larvae, and they speculated that reproductive chimeras could exist in nature, a concept first proposed by Mukai (1992) for the freshwater sponge, Ephydatia muelleri. The occurrence of interspecific sponge chimerism and somatic hybridization (i.e., hybridomas) in nature could affect the interpretation of phylogenetic relationships based on DNA sequencing of small subsamples of sponges. Experimental sponge hybridomas and chimeras are useful models for testing hypotheses related to interpretation of phylogenetic relationships based on molecular analyses of sponges, as well as different evolutionary scenarios, such as a proclivity for natural hybridization, co-evolution of chimeric or symbiotic partners, and molecular mimicry.

Acknowledgments The authors thank Drs Amy Wright, Esther Guzman, Susan Sennett, James Grasela (Florida Atlantic University), Drs Rene Wijffels and Klaske Schippers (Wageningen University, The Netherlands), Dr Felicia Coleman (Florida State University), Dr Christine Morrow (Queen’s University, Belfast), and Dr Nicole Boury-Esnault (CNRS, Marseille) for their insights into the development and application of sponge hybridomas and for their advice related to sponge systematics and phylogenetics. We thank Tara Pitts and Jennifer Grima (Florida Atlantic University) for assistance with laboratory experiments, Drs Jose Lopez and Andia Chavez-Fonnegra (Nova Southeastern University) for assistance with field collections, and Dr James Masterson (Florida Atlantic University) for assistance with preparation of figures. This is Harbor Branch Oceanographic Institute contribution number 1888.

Funding This work was supported by the National Science Foundation [grant numbers 0829791 to S.A.P. as co-PI, 0829986 to M.C.D. as co-PI], the National Oceanic and Atmospheric Administration, Florida Sea Grant College Program [grant number R/LRMB-25 to S.A.P.], and the Gertrude E. Skelly Charitable Foundation summer internship program [to A.J. and J.P.].

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