CELLULAR & MOLECULAR BIOLOGY LETTERS http://www.cmbl.org.pl Received: 07 November 2014 Final form accepted: 19 January 2015 Published online: 28 February 2015
Volume 20 (2015) pp 117-129 DOI: 10.1515/cmble-2015-0003 © 2014 by the University of Wrocław, Poland
Short communication ONCOGENE-DEPENDENT SURVIVAL OF HIGHLY TRANSFORMED CANCER CELLS UNDER CONDITIONS OF EXTREME CENTRIFUGAL FORCE – IMPLICATIONS FOR STUDIES ON EXTRACELLULAR VESICLES TAE HOON LEE, SHILPA CHENNAKRISHNAIAH and JANUSZ RAK* Montreal Children’s Hospital Research Institute, 4060 Ste Catherine West, Montreal, Quebec, H3Z 2Z3, Canada Abstract: Extracellular vesicles (EVs), including exosomes, are a subject of intense interest due to their emission by cancer cells and role in intercellular communication. Earlier reports suggested that oncogenes, such as RAS, MET or EGFR, drive cellular vesiculation. Interestingly, these oncogenes may also traffic between cells using the EV-mediated emission and uptake processes. One of the main tools in the analysis of EVs are ultracentrifugation protocols designed to efficiently separate parental cells from vesicles through a sequence of steps involving increasing g-force. Here we report that ultracentrifugationonly EV preparations from highly transformed cancer cells, driven by the overexpression of oncogenic H-ras (RAS-3) and v-src (SRC-3), may contain clonogenic cancer cells, while preparations of normal or less aggressive human cell lines are generally free from such contamination. Introduction of a filtration step eliminates clonogenic cells from the ultracentrifugate. The survival of RAS-3 and SRC-3 cells under extreme conditions of centrifugal force (110,000 g) is oncogene-induced, as EV preparations of their parental non-tumourigenic cell line (IEC-18) contain negligible numbers of clonogenic cells. Moreover, treatment of SRC-3 cells with the SRC inhibitor (PP2) markedly reduces the presence of such cells in the unfiltered ultracentrifugate. These observations enforce the notion that EV preparations require careful filtration steps, especially in the case of material produced by highly transformed cancer cell types. * Author for correspondence. Email: [email protected] Abbreviations used: EGFR/EGFRvIII – epidermal growth factor receptor; EV – extracellular vesicle; FBS – fetal bovine serum; gDNA – genomic DNA; IEC – intestinal epithelial cell; NHA – normal human astrocyte; PC – post-centrifugation; PES – polyethersulfone; TEM – transmission electron microscopy
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We also suggest that oncogenic transformation may render cells unexpectedly resistant to extreme physical forces, which may affect their biological properties in vivo. Keywords: Extracellular vesicle, H-ras, v-src, Ultracentrifugation, PP2, Hyperacceleration, Filtration, TEM INTRODUCTION There is a growing interest in ‘unconventional’ constituents of the cellular secretome, including various subsets of extracellular vesicles (EVs) [1-3]. EVs are membrane-derived external quasi-organelles that mediate the selective release of cellular content into the surrounding microenvironment. The diverse and still poorly understood biogenetic pathways drive formation of EVs with different sizes and properties, most notably a fraction of small vesicles (30 – 100 nm in diameter) often described as exosomes [2]. Exosomes have been implicated as regulators of hematopoietic, immune and inflammatory processes, where their rich sources include reticulocytes, mast cells and antigen presenting cells that release antigens, receptors, bioactive proteins and nucleic acids as vesicular cargo [4-7]. Isolation and characterization of exosomes involves filtration, precipitation and immunoaffinity capture-based methods [1], but is most often achieved by protocols based on differential centrifugation of cell culture supernatant or biofluids. In this case, the cells are separated at the lower centrifugal force (usually approximately 400 g), and the EV fractions are sedimented from the remaining fluid phase by several ultracentrifugation steps, of which the force of 110,000 g is used to isolate exosome-sized vesicles from larger ectosomes [8, 9]. This approach is believed to reliably separate cellular material from exosomes due to the application of drastically different centrifugal forces, which normal cells are believed to be unable to withstand. While these methods have been extensively validated for exosomes from immune and hematopoietic cells [8], similar approaches have recently been used to satisfy the intense interest in EV production by cancer cells. In this case, exosomes have been implicated in processes of intercellular communication, cancer progression, invasion, angiogenesis, metastasis and emission of cancer biomarkers [10-15]. One mechanistic link in this regard is the ability of oncogenic pathways to impact biogenesis and cargo of tumour-derived exosomes [16]. Indeed, we have recently demonstrated that the expression of several oncogenic forms of epidermal growth factor receptor (EGFR/EGFRvIII) stimulate vesiculation of cancer cells, leading to the extracellular emission of several bioactive and insoluble molecular entities, including the oncogenic EGFR protein and mRNA. Notably, the uptake of this material by indolent cells leads to transformation-like changes in their phenotype [13, 17-18]. Similar events have also been described in the case of several other oncogenes [16]. In particular, transformation of intestinal epithelial cells (IEC-18) by
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exogenous introduction of the oncogenic H-ras or v-src leads to the emission of bioactive exosome-like EVs containing the respective oncogenic cargo, including the genomic double-stranded H-ras sequences [19], which have also been described in other cellular contexts [20-23]. The presence of genomic DNA in a fraction of exosome-sized EVs is surprising, as such material is typically associated with cellular nuclei or apoptotic bodies, and it raises questions as to what is the nature of the material isolated as exosomal fraction from cancer cell supernatants through standard ultracentrifugation protocols. Here we show that ultracentrifugation is not sufficient to reliably isolate exosome-like EVs from certain cancer cell population. This is because ras- and src-mediated transformation enables substantial numbers of cells to enter and remain viable within the standard exosomal pellets obtained under vacuum and ultracentrifugation at 110,000 g. This was not observed in EV preparations of corresponding non-transformed cells or of less aggressive cellular populations, and it diminished in the presence of the oncogene inhibitor. This observation suggests a link between oncogenic transformation and the unusual resistance of cancer cells to extreme physical forces applied during ultracentrifugation. While the biological relevance of this property requires further study, we suggest that filtration is an essential step in EV preparation protocols involving highly transformed cancer cells. MATERIALS AND METHODS Cell culture and treatment conditions The following cell lines were used in the study: IEC-18 – non-tumourigenic, immortalized rat intestinal epithelial cell line; RAS-3 – tumourigenic, clonal IEC-18 subline that has been transfected with a V12 mutant, activated c-H-ras human oncogene; SRC-3 – tumourigenic, clonal variant of IEC-18 cells that has been transfected with the v-src oncogene; A431 – tumourigenic, human epidermoid carcinoma cell line harbouring oncogenic EGFR; NHA – nontumourigenic, normal human astrocyte cell line immortalized using human telomerase (hTERT), a gift from Dr. Guha, University of Toronto; U373P (or U373) – parental non-tumourigenic/indolent human glioblastoma cell line; U373vIII – tumourigenic variant of U373P cells, harbouring a constitutively active EGFRvIII oncogene [17]. IEC-18, RAS-3 and SRC-3 cells were grown as previously described [24]. A431, NHA and U373P cells were maintained in DMEM supplemented with 10% FBS (#080-150, Wisent Bioproducts) and 1% Penn/Strep (#15140, GIBCO). U373vIII cells were maintained under similar conditions with addition of 10% Tetracycline-free FBS (#081-150, Wisent Bioproducts). For experiments involving EV isolation, the media were supplemented with 10% EV-depleted FBS (ultracentrifugation at 150,000 g for 5 hours). Inhibition of SRC activity in SRC-3 cells was achieved by treating them with 20 µM PP2 compound (#P0042, Sigma) [25]. RAS-3-derived cell lines were developed by expanding the selected clones that survived
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ultracentrifugation, designated as PC1RAS-3, PC2RAS-3 and PC3RAS-3, and cultured as indicated. Isolation of extracellular vesicles EVs were obtained by ultracentrifugation either as previously described, or by a simplified protocol involving a slight modification in the number of centrifugation steps [17, 19]. Briefly, conditioned media were first centrifuged for 10 minutes at 400 g to remove cells and debris, followed by filtration through a 0.22 µm PES filter (#565-0020, Thermo Fisher Scientific). The filtrate was then ultracentrifuged for 1 hour at 110,000 g to isolate the pure EV fraction. In some cases, cells themselves were subjected to an extended ultracentrifugation, as indicated. Transmission electron microscopy (TEM) Cells and isolated EVs were washed once with 0.1M sodium cacodylate buffer, pH 7.4 and then fixed with 2.5% glutaraldehyde in the same buffer. The fixed samples were subjected to either ultramicrotomy for processing of monolayer cells, or whole mount negative staining for capturing of all EVs in the fixative. Tecnai 12 BioTwin 120 kV TEM was used to capture images. Clonogenic cell survival Following ultracentrifugation of the conditioned media at 110,000 g with or without filtration, the pellets were resuspended in complete media and cultured for 7 days prior to fixation with 10% formalin. Dishes were washed with distilled water and then stained for 20 minutes with 0.2% crystal violet in 20% methanol. After another wash with distilled water, images of clonal colonies were taken and their numbers quantified manually in the image of the entire well. MTS proliferation assay Viability or time-dependent increase in metabolic activity of growing cells was assessed using MTS assays (#G3580, Promega). Briefly, the indicated cells were plated in 96 well plates, grown for 1, 2 and 3 days, and processed according to the manufacturer’s protocol. To assess the recovery of viable cells following mechanical stress, 1,000 cells were ultracentrifuged directly at 110,000 g and MTS assay was performed at days 1, 2 and 3, as described. Data analysis Colony formation assays were performed at least twice with similar results. Experiments involving the same cell line across different studies (e.g. RAS-3) were done independently from one another. The statistical differences of resulting clonogenic recovery counts was analysed using computerised one-way ANOVA test, where comparisons were made to the reference cell line, as indicated. MTS proliferation assay results (N=4) were processed for statistical significance using the non-parametrical Mann-Whitney U test (Wilcoxon rank sum test). The threshold p-value of 0.05 was taken as a measure of significance.
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RESULTS AND DISCUSSION H-ras expressing cancer cells survive forces of ultracentrifugation In our prior studies on the cargo of EVs produced by cancer cells, we observed that this material contains bioactive oncogenes in a form of intact oncoproteins, mRNA and genomic DNA (gDNA) sequences [17-19]. This cargo can be recovered using either a complete EV preparation protocol involving 0.22 µm filtration of the culture supernatant, followed by ultracentrifugation at 110,000 g [19], or through a simplified (high yield) protocol containing pre-clearing and ultracentrifugation step alone (Fig. 1A). Thus, in the case of highly tumourigenic rat intestinal cancer cells harbouring the human mutant H-ras oncogene (RAS-3), transmission electron microscopy (TEM) reveals structural perturbations near the cellular plasma membrane and the presence of multiple exosome-like EVs, approximately 100 nm in diameter, in the ultracentrifugate of the cultured medium, with no evidence of debris or contaminating cells (Fig. 1B). However, the ample presence of the double-stranded gDNA in ultracentrifugates of RAS-3 conditioned media prompted us to examine whether, in spite of convincing TEM imagery, this material may contain rare viable RAS-3 cells that evade standard centrifugal pre-clearing (400 g). To do so, ultracentrifugation pellets of RAS-3 conditioned media were resuspended in medium and plated to test for the possible presence of tumour cell colonies (Fig. 1A). Interestingly, this assay revealed multiple colonies of clonogenic and proliferative cancer cells in spite of the extreme centrifugal force applied to them during the 1 hour ultracentrifugation in vacuum and at 110,000 g force (Fig. 1C). All such growth was eliminated when the media was passed through a 0.22 µm filter before ultracentrifugation, as per our standard protocol (Fig. 1AD), which continues to yield exosomal gDNA sequences [19]. These observations are important for at least two reasons. First, they enforce the notion that isolation of pure EVs produced by cancer cells should occur with inclusion of filtration steps to eliminate the rare cancer cells that may enter these preparations in spite of centrifugal pre-clearing. This is especially important in the case of bioassays aimed to demonstrate the EV-mediated transfer of biological properties from cancer to normal cells, as the presence of viable cancer cells could conceivably mimic such a transfer. Second, it is intriguing that at least some RAS-3 cells were able to survive extreme and prolonged (1 hour) forces of vacuum ultracentrifugation and retain their clonogenic potential under such harsh conditions. RAS-3 cells surviving ultracentrifugation exhibit sustained clonogenic potential To explore if survival under ultracentrifugation conditions represents a feature of some or all cells within the clonal RAS-3 population, we isolated individual colonies surviving this procedure and established three post-centrifugation (PC) clonal cellular sublines (PC variants – PC1RAS-3, PC2RAS-3 and PC3RAS-3).
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Fig. 1. Survival of H-ras-transformed cancer cells under conditions of vacuum ultracentrifugation. A – Schematic diagram illustrating EV preparations using the filtration (left) and no filtration (right) methods, as well as the workflow of the analysis presented. B – Vesiculation (arrows) observed at the surface of H-ras-transformed RAS-3 epithelial cancer cells. Transmission electron microscopy (TEM) images were taken at 23k (cells) and 49k (filtered EVs) magnifications, respectively. C – Unfiltered EV ultracentrifugate contains clonogenic RAS-3 cells. D – Filtered EV ultracentrifugate contains no clonogenic cells. E – The number of colonies recovered from the ultracentrifugates of conditioned
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media of RAS-3 and their variant cell lines (PC1RAS-3 – PC3RAS-3). These cells were expanded from clonal colonies grown previously from ultracentrifugates of RAS-3 conditioned media (p-values calculated using computerized one-way ANOVA versus RAS-3 cells). F – The viability or time-dependent increase in metabolic activity of RAS-3 and its variant cell lines were measured using MTS and found largely comparable except in PC1RAS-3 variants (N=4; *Wilcoxon rank sum test < 0.05). G – The viability or timedependent increase in metabolic activity of RAS-3 and its variant cell lines were measured after direct ultracentrifugation (1,000 cells; 110,000 g for 1 hour) by MTS and found largely comparable except in PC1RAS-3 variants (N=4; *Wilcoxon rank sum test < 0.05).
Equal number of these cells and their RAS-3 parental population were plated to generate conditioned media and this material was subjected to another round of ultracentrifugation and plating for clonogenic recovery of cancer cells. Notably, in the case of two out of three PC cell lines, the number of colonies recovered from such ultracentrifugate was higher, in one case significantly, compared to that of parental RAS-3 cells (Fig 1E). We also compared the viability of PC variants either by testing cells present in the culture conditioned medium (Fig. 1F) or when they were directly subjected to ultracentrifugation forces (Fig. 1G). These experiments, involving MTS assays, did not reveal consistent differences in the state or time dependent changes of metabolic activity (which may reflect growth or demise) between PC cell lines relative to their parental RAS-3 counterparts, even though one cell line (PC1RAS-3) had a somewhat higher metabolic rate and exhibited a more spindle morphology (data not shown). Pelleting trypsinized cells by vacuum ultracentrifugation at 110,000 g, once again, confirmed that both RAS-3 cells and their PC variants can withstand such conditions and retain their timedependent increase in metabolic activity, as measured by the MTS assay (Fig. 1G). This remained unchanged even after 3 hours of continuous ultracentrifugation (data not shown). Collectively, these observations suggest that RAS-3 cells are remarkably resistant to extreme centrifugal forces. Notably, the clonogenic recovery appears to differ between subpopulations of the clonal RAS-3 cell line, which may reveal the existence of cellular subsets (possibly new mutants) more resistant to this treatment. This raises the question as to whether such heterogeneity exists between other transformed and normal cells. Differential recovery of clonogenic cells from ultracentrifugates of transformed and non-transformed cell lines To explore whether the ability to withstand ultracentrifugation is specific to RAS-3 cells or common for cancer cells, we collected conditioned media from several human cell lines, including epidermoid and glioblastoma cells harbouring oncogenic EGFR (A431 and U373vIII cells), indolent but genetically transformed glioma cells (U373P), and immortalized but non-tumourigenic (normal) human astrocytes (NHA). Surprisingly, in contrast to RAS-3 cells, we were unable to consistently recover viable cells from centrifugates of any of these cell lines after their exposure to 110,000 g force, with a possible
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(paradoxical) exception of NHA cells, which, albeit inconsistently, generated individual colonies under such conditions (Table 1). It is of note that while A431 and U373vIII are tumourigenic, their relative in vivo aggressiveness is far less pronounced than that of RAS-3 cells, which give rise to a highly malignant and angiogenic tumours, and are resistant to anoikis and other insults [24, 26]. Therefore, it is possible that preferential survival of RAS-3 cells under extreme centrifugal forces is linked to some features of their malignant phenotype. Table 1. Clonogenic recovery of viable non-tumourigenic and tumourigenic cells from ultracentrifugates of culture conditioned media. Indicated cells with different tissue origin, aggressiveness and type of oncogenic transformation were cultured, conditioned medium collected and subjected to ultracentrifugation without filtration, as in the simplified protocol for EV preparation (Fig 1A). Pooled data from 6 independent experiments are shown, with numbers indicating colony counts from individual replicate wells. Of note, high level of transformation achieved by the expression of oncogenic H-ras or v-src rendered IEC-18 intestinal epithelial cells resistant to considerable centrifugal force. Nontransformed cells (IEC-18, NHA), indolent (U373P) and moderately transformed cancer cells (A431, U373vIII) exhibited a comparable, overall very low ability to yield viable cells in ultracentrifugates. P-values as indicated (one-way ANOVA), for comparisons with reference cell lines (* to ****, as indicated). Cells A431
Characteristics (origin and type of transformation)
NHA
Tumourigenic EGFR-driven carcinoma (human) Immortalized astrocytes (human)
U373P
Recovered P–value colony counts ANOVA (independent repeats) 3, 1 0.0014* 12, 1
0.0365*
Indolent glioma (human)
0, 1
Tumourigenic EGFRvIII-driven glioma (human) RAS-3 Highly aggressive H-ras-driven IEC-18derived carcinoma (rat) SRC-3 Highly aggressive v-src-driven IEC-18derived carcinoma (rat) SRC-3 + PP2 SRC-3 cells pre-treated with SRC inhibitor (PP2) IEC-18 Immortalized intestinal epithelium (rat)
0, 1 129, 82, 62, 157
0.0301* 0.3908**** 0.0301* 0.3907**** NA*
143, 96, 68, 121
0.9858*(NS)
20, 10
0.0196*** 0.0007** 0.0031*
U373vIII
10, 0, 3, 2
* - vs RAS-3; ** - vs IEC-18; *** - vs SRC-3, **** - vs NHA
Oncogenic transformation as a determinant of clonogenic cancer cell recovery upon ultracentrifugation RAS-3 cells are derivatives of the phenotypically normal, anoikis-sensitive and non-tumourigenic, but immortalized and clonogenic, rat intestinal epithelial cell line (IEC-18). From these cells, several highly aggressive tumourigenic variants were established following a single step transfection with H-ras or v-src
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oncogenes [24], as exemplified by RAS-3 and SRC-3 cells, respectively. Given the appreciable clonogenic recovery of viable cells from RAS-3 ultracentrifugates, it is reasonable to ask whether this property is cell line/typespecific or linked to a defined oncogenic event readily traceable in this simple model system. To address this question, IEC-18, RAS-3 and SRC-3 cells were used to generate conditioned media, which were subjected to 110,000 g ultracentrifugation and colony formation assays. Notably, the parental IEC-18 cells revealed negligible numbers of colonies following this procedure, while both RAS-3 and SRC-3 cells gave rise to ample clonogenic growth (Table 1). In this regard, the existence of specific and potent inhibitors of the SRC oncoprotein, such as PP2, offers an opportunity to examine whether the clonogenic survival of SRC-3 cells under centrifugal forces continues to depend on the transforming effect of the v-src oncogene. This appears to be the case, as conditioned medium of PP2-treated (but viable) SRC-3 cells generated reduced numbers of clonal colonies upon ultracentrifugation (Table 1). These results suggest that the ability to withstand centrifugal forces and contribute viable cells to the EV pellet is a function of oncogenic transformation, at least as it applies to cells exhibiting high expression of mutant H-ras or v-src oncogenes. In conclusion, our findings suggest that certain forms of oncogenic transformations confer cellular resistance to extreme physical forces. We serendipitously identified this influence while exploring protocols of ultracentrifugation involving centrifugal forces up to 110,000 g. This has several implications for techniques aimed at isolating EVs from cancer cell cultures (and biofluids) using simplified ultracentrifugation protocols. While such protocols may be effective in the case of normal cells and provide a high yield of EVs, our exploration of their use in the case of highly transformed cancer cells raises a cautionary note. The presence of viable and clonogenic cancer cells is not eliminated by the standard pre-clearing or extreme centrifugal forces, and may impact assays aimed at detecting various EV-specific molecular materials and influence the outcomes of relevant bioassays. These risks could be effectively addressed by maintaining a filtration step as an integral part of these experiments, one that effectively eliminates cellular contamination, but permits detection of EV-associated oncogenic cargo, including gDNA [19]. Unexpectedly, our study revealed a differential impact of the level and nature of oncogenic transformation on the survival of cancer cells under extreme physical challenges, as exemplified by prolonged vacuum ultracentrifugation. We observed that high levels of oncogenic H-ras and v-src expression, but not oncogenic EGFR, enable such survival, an observation that warrants further study as to how these respective oncogenes influence mechanosensory and physical properties of cancer cells. We and others have previously documented the resistance of RAS-3 and SRC-3 cells to detachment-induced cell death (anoikis), in contrast to their non-transformed IEC-18 counterparts [26]. This could be attributed to the relative uniqueness of signalling properties associated with the oncogenic RAS [27], and its impact on cytoskeletal organization [28-30].
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Of note is the fact that forces exerted on cells within the physical microenvironment are increasingly recognized as biologically important cues in cancer, including the cellular phenotype, cell shape, polarity and selection of stem cell populations [31-34]. The impact of the RAS pathway on mechanosensing and mechanical resistance is therefore of utmost interest. While forces applied to the cells at 110,000 g ultracentrifugation are artificial and likely exceed the conditions ordinarily encountered in vivo, cellular responses they elicit may signify the ability of cancer cells to survive relevant mechanical stresses, such as during the process of metastasis, invasive crossing of tissue barriers and expansion in confined tissue microenvironments, such as brain or bone. This includes behaviour of cells under conditions of movement, including rarely discussed circumstances such as aerial or space travel [35]. While few studies explored these questions in eukaryotic cells, certain bacteria have been documented to survive hyperaccelerative forces in excess of 400,000 g [36, 37], a finding that raises questions as to possible commonalities of these biological mechanisms and biomechanical principles to those operative in eukaryotic and diseased (cancer) cells. Implications of physical forces in human pathology remain to be thoroughly investigated. Acknowledgements. We thank our McGill colleagues J. Mui and L. Mongeon for their assistance in electron microscopy. This project was supported by a grant from the Canadian Institutes of Health Research MOP 11119 and 133424 (J.R.). J.R. is the Jack Cole Chair in Paediatric Haematology/Oncology. Support was also provided (S.C.) by MELS (Fonds de recherche du Quebec - Nature et technologies) and R. Tomlinson studentships. Infrastructure support was provided by the Fonds de la recherche en santé du Québec. REFERENCES 1. Lee, T.H., D’asti, E., Magnus, N., Al-Nedawi, K., Meehan, B. and Rak, J. Microvesicles as mediators of intercellular communication in cancer–the emerging science of cellular ‘debris’. Semin. Immunopathol. 33 (2011) 455-467. DOI: 10.1007/s00281-011-0250-3. 2. Thery, C. Exosomes: secreted vesicles and intercellular communications. F1000 Biol. Rep. 3 (2011) 15. DOI: 10.3410/B3-15. 3. Gyorgy, B., Szabo, T.G., Pasztoi, M., Pal, Z., Misjak, P., Aradi, B., Laszlo, V., Pallinger, E., Pap, E., Kittel, A., Nagy, G., Falus, A. and Buzas, E.I. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol. Life Sci. 68 (2011) 2667-2688. DOI: 10.1007/s00018011-0689-3. 4. Johnstone, R.M. Exosomes biological significance: A concise review. Blood Cells Mol. Dis. 36 (2006) 315-321. 5. Thery, C., Ostrowski, M. and Segura, E. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 9 (2009) 581-593. DOI: 10.1038/nri2567.
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6. Zitvogel, L., Regnault, A., Lozier, A., Wolfers, J., Flament, C., Tenza, D., Ricciardi-Castagnoli, P., Raposo, G. and Amigorena, S. Eradication of established murine tumours using a novel cell-free vaccine: dendritic cellderived exosomes. Nat. Med. 4 (1998) 594-600. 7. Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J.J. and Lotvall, J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9 (2007) 654-659. 8. Thery, C., Amigorena, S., Raposo, G. and Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. 3 (2006) 3.22. DOI: 10.1002/0471143030. 9. Bianco, F., Perrotta, C., Novellino, L., Francolini, M., Riganti, L., Menna, E., Saglietti, L., Schuchman, E.H., Furlan, R., Clementi, E., Matteoli, M. and Verderio, C. Acid sphingomyelinase activity triggers microparticle release from glial cells. EMBO J. 28 (2009) 1043-1054. DOI: 10.1038/emboj.2009.45. 10. Luga, V., Zhang, L., Viloria-Petit, A.M., Ogunjimi, A.A., Inanlou, M.R., Chiu, E., Buchanan, M., Hosein, A.N., Basik, M. and Wrana, J.L. Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 151 (2012) 1542-1556. DOI: 10.1016/j.cell.2012.11.024. 11. Peinado, H., Aleckovic, M., Lavotshkin, S., Matei, I., Costa-Silva, B., Moreno-Bueno, G., Hergueta-Redondo, M., Williams, C., Garcia-Santos, G., Ghajar, C., Nitadori-Hoshino, A., Hoffman, C., Badal, K., Garcia, B.A., Callahan, M.K., Yuan, J., Martins, V.R., Skog, J., Kaplan, R.N., Brady, M.S., Wolchok, J.D., Chapman, P.B., Kang, Y., Bromberg, J. and Lyden, D. Melanoma exosomes educate bone marrow progenitor cells toward a prometastatic phenotype through MET. Nat. Med. 18 (2012) 883-891. DOI: 10.1038/nm.2753. 12. Poste, G. and Nicolson, G.L. Arrest and metastasis of blood-borne tumour cells are modified by fusion of plasma membrane vesicles from highly metastatic cells. Proc. Natl. Acad. Sci. USA 77 (1980) 399-403. 13. Skog, J. Wurdinger, T., van Rijn, S., Meijer, D.H., Gainche, L., Curry, Jr. W.T., Carter, B.S., Krichevsky, A.M. and Breakefield, X.O. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10 (2008) 1470-1476. DOI: 10.1038/ncb1800. 14. Christianson, H.C., Svensson, K.J., van Kuppevelt, T.H., Li, J.P. and Belting, M. Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity. Proc. Natl. Acad. Sci. USA 110 (2013) 17380-17385. DOI: 10.1073/pnas.1304266110. 15. Taylor, D.D. and Gercel-Taylor, C. MicroRNA signatures of tumour-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol. Oncol. 110 (2008) 13-21. DOI: 10.1016/j.ygyno.2008.04.033.
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16. D’Asti, E., Garnier, D., Lee, T.H., Montermini, L., Meehan, B. and Rak, J. Oncogenic extracellular vesicles in brain tumour progression. Front. Physiol. 3 (2012) 1-15. DOI: 10.3389/fphys.2012.00294. eCollection 2012. 17. Al-Nedawi, K., Meehan, B., Micallef, J., Lhotak, V., May, L., Guha, A. and Rak, J. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat. Cell Biol. 10 (2008) 619-624. DOI: 10.1038/ncb1725. 18. Al-Nedawi K., Meehan, B., Kerbel, R.S., Allison, A.C. and Rak, J. Endothelial expression of autocrine VEGF upon the uptake of tumourderived microvesicles containing oncogenic EGFR. Proc. Natl. Acad. Sci. USA 106 (2009) 3794-3799. DOI: 10.1073/pnas.0804543106. 19. Lee, T.H., Chennakrishnaiah, S., Audemard, E., Montermini, L., Meehan, B. and Rak, J. Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells. Biochem. Biophys. Res. Commun. 451 (2014) 295-301. DOI: 10.1016/j.bbrc.2014.07.109. 20. Balaj, L., Lessard, R., Dai, L., Cho, Y.J., Pomeroy, S.L., Breakefield, X.O. and Skog, J. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat. Commun. 2 (2011). DOI: 10.1038/ncomms1180. 21. Holmgren, L., Szeles, A., Rajnavolgyi, E., Folkman, J., Klein, G., Emberg, I. and Falk, K.I. Horizontal transfer of DNA by the uptake of apoptotic bodies. Blood 93 (1999) 3956-3963. 22. Thakur, B.K., Zhang, H., Becker, A., Matei, I., Huang, Y., Costa-Silva, B., Zheng, Y., Hoshino, A., Brazier, H., Xiang, J., Williams, C., RodriguezBarrueco, R., Silva, J.M., Zhang, W., Hearn, S., Elemento, O., Paknejad, N., Manova-Todorova, K., Welte, K., Bromberg, J., Peinado, H. and Lyden, D. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 24 (2014) 766-769. DOI: 10.1038/cr.2014.44. 23. Kahlert, C., Melo, S.A., Protopopov, A., Tang, J., Seth, S., Koch, M., Zhang, J., Weitz, J., Chin, L., Futreal, A. and Kalluri, R. Identification of doublestranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J. Biol. Chem. 289 (2014) 3869-3875. DOI: 10.1074/jbc.C113.532267. 24. Rak, J., Mitsuhashi, Y., Bayko, L., Filmus, J., Shirasawa, S., Sasazuki, T. and Kerbel, R.S. Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumour angiogenesis. Cancer Res. 55 (1995) 4575-4580. 25. Kong, L., Deng, Z., Shen, H. and Zhang, Y. Src family kinase inhibitor PP2 efficiently inhibits cervical cancer cell proliferation through down-regulating phosphor-Src-Y416 and phosphor-EGFR-Y1173. Mol. Cell. Biochem. 348 (2011) 11-19. DOI: 10.1007/s11010-010-0632-1. 26. Rak, J., Mitsuhashi, Y., Erdos, V., Huang, S.N., Filmus, J. and Kerbel, R.S Massive programmed cell death in intestinal epithelial cells induced by
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three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression. J. Cell Biol. 131 (1995) 1587-1598. Karnoub, A.E. and Weinberg, R.A. Ras oncogenes: split personalities. Nat. Rev. Mol. Cell Biol. 9 (2008) 517-531. DOI: 10.1038/nrm2438. Cox, A.D., Fesik, S.W., Kimmelman, A.C., Luo, J. and Der, C.J. Drugging the undruggable RAS: Mission possible? Nat. Rev. Drug Discov. 13 (2014) 828-851. DOI: 10.1038/nrd4389. Sumita, K., Yoshino, H., Sasaki, M., Majd, N., Kahoud, E.R., Takahashi, H., Takeuchi, K., Kuroda, T., Lee, S., Charest, P.G., Takeda, K., Asara, J.M., Firtel, R.A., Anastasiou, D. and Sasaki, A.T. Degradation of activated K-Ras orthologue via K-Ras-specific lysine residues is required for cytokinesis. J. Biol. Chem. 289 (2014) 3950-3959. DOI: 10.1074/jbc.M113.531178. Commisso, C., Davidson, S.M., Soydaner-Azeloglu, R.G., Parker, S.J., Kamphorst, J.J., Hackett, S., Grabocka, E., Nofal, M., Drebin, J.A., Thompson, C.B., Rabinowitz, J.D., Metallo, C.M., Vander Heiden, M,G. and Bar-Sagi, D. Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature 497 (2013) 633-637. DOI: 10.1038/nature12138. Vogel, V. and Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7 (2006) 265-275. Milsom, C. and Rak, J. Regulation of tissue factor and angiogenesis-related genes by changes in cell shape. Biochem. Biophys. Res. Commun. 337 (2005) 1267-1275. Ingber, D.E. Tensegrity I. Cell structure and hierarchical systems biology. J. Cell Sci. 116 (2003) 1157-1173. Liu, J., Tan, Y., Zhang, H., Zhang, Y., Xu, P., Chen, J., Poh, Y.C., Tang, K., Wang, N. and Huang, B. Soft fibrin gels promote selection and growth of tumourigenic cells. Nature Mat. 11 (2012) 734-742. DOI: 10.1038/nmat3361. Guignandon, A., Faure, C., Neutelings, T., Rattner, A., Mineur, P., Linossier, M., Laroche, N., Lambert, C., Deroanne, C., Nusgens, B., Demets, R., Colige, A. and Vico, L. Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells. FASEB J. 28 (2014) 4077-4087. Klaus, D., Simske, S., Todd, P. and Stodieck, L. Investigation of space flight effects on Escherichia coli and a proposed model of underlying physical mechanisms. Microbiology 143 (1997) 449-455. Deguchi, S., Shimoshige, H., Tsudome, M., Mukai, S.A., Corkery, R.W., Ito, S. and Horikoshi, K. Microbial growth at hyperaccelerations up to 403,627 * g. Proc. Natl. Acad. Sci. USA 108 (2011) 7997-8002. DOI: 10.1073/pnas.1018027108.
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Volume 20 (2015) pp 117-129 DOI: 10.1515/cmble-2015-0003 © 2014 by the University of Wrocław, Poland
Short communication ONCOGENE-DEPENDENT SURVIVAL OF HIGHLY TRANSFORMED CANCER CELLS UNDER CONDITIONS OF EXTREME CENTRIFUGAL FORCE – IMPLICATIONS FOR STUDIES ON EXTRACELLULAR VESICLES TAE HOON LEE, SHILPA CHENNAKRISHNAIAH and JANUSZ RAK* Montreal Children’s Hospital Research Institute, 4060 Ste Catherine West, Montreal, Quebec, H3Z 2Z3, Canada Abstract: Extracellular vesicles (EVs), including exosomes, are a subject of intense interest due to their emission by cancer cells and role in intercellular communication. Earlier reports suggested that oncogenes, such as RAS, MET or EGFR, drive cellular vesiculation. Interestingly, these oncogenes may also traffic between cells using the EV-mediated emission and uptake processes. One of the main tools in the analysis of EVs are ultracentrifugation protocols designed to efficiently separate parental cells from vesicles through a sequence of steps involving increasing g-force. Here we report that ultracentrifugationonly EV preparations from highly transformed cancer cells, driven by the overexpression of oncogenic H-ras (RAS-3) and v-src (SRC-3), may contain clonogenic cancer cells, while preparations of normal or less aggressive human cell lines are generally free from such contamination. Introduction of a filtration step eliminates clonogenic cells from the ultracentrifugate. The survival of RAS-3 and SRC-3 cells under extreme conditions of centrifugal force (110,000 g) is oncogene-induced, as EV preparations of their parental non-tumourigenic cell line (IEC-18) contain negligible numbers of clonogenic cells. Moreover, treatment of SRC-3 cells with the SRC inhibitor (PP2) markedly reduces the presence of such cells in the unfiltered ultracentrifugate. These observations enforce the notion that EV preparations require careful filtration steps, especially in the case of material produced by highly transformed cancer cell types. * Author for correspondence. Email: [email protected] Abbreviations used: EGFR/EGFRvIII – epidermal growth factor receptor; EV – extracellular vesicle; FBS – fetal bovine serum; gDNA – genomic DNA; IEC – intestinal epithelial cell; NHA – normal human astrocyte; PC – post-centrifugation; PES – polyethersulfone; TEM – transmission electron microscopy
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We also suggest that oncogenic transformation may render cells unexpectedly resistant to extreme physical forces, which may affect their biological properties in vivo. Keywords: Extracellular vesicle, H-ras, v-src, Ultracentrifugation, PP2, Hyperacceleration, Filtration, TEM INTRODUCTION There is a growing interest in ‘unconventional’ constituents of the cellular secretome, including various subsets of extracellular vesicles (EVs) [1-3]. EVs are membrane-derived external quasi-organelles that mediate the selective release of cellular content into the surrounding microenvironment. The diverse and still poorly understood biogenetic pathways drive formation of EVs with different sizes and properties, most notably a fraction of small vesicles (30 – 100 nm in diameter) often described as exosomes [2]. Exosomes have been implicated as regulators of hematopoietic, immune and inflammatory processes, where their rich sources include reticulocytes, mast cells and antigen presenting cells that release antigens, receptors, bioactive proteins and nucleic acids as vesicular cargo [4-7]. Isolation and characterization of exosomes involves filtration, precipitation and immunoaffinity capture-based methods [1], but is most often achieved by protocols based on differential centrifugation of cell culture supernatant or biofluids. In this case, the cells are separated at the lower centrifugal force (usually approximately 400 g), and the EV fractions are sedimented from the remaining fluid phase by several ultracentrifugation steps, of which the force of 110,000 g is used to isolate exosome-sized vesicles from larger ectosomes [8, 9]. This approach is believed to reliably separate cellular material from exosomes due to the application of drastically different centrifugal forces, which normal cells are believed to be unable to withstand. While these methods have been extensively validated for exosomes from immune and hematopoietic cells [8], similar approaches have recently been used to satisfy the intense interest in EV production by cancer cells. In this case, exosomes have been implicated in processes of intercellular communication, cancer progression, invasion, angiogenesis, metastasis and emission of cancer biomarkers [10-15]. One mechanistic link in this regard is the ability of oncogenic pathways to impact biogenesis and cargo of tumour-derived exosomes [16]. Indeed, we have recently demonstrated that the expression of several oncogenic forms of epidermal growth factor receptor (EGFR/EGFRvIII) stimulate vesiculation of cancer cells, leading to the extracellular emission of several bioactive and insoluble molecular entities, including the oncogenic EGFR protein and mRNA. Notably, the uptake of this material by indolent cells leads to transformation-like changes in their phenotype [13, 17-18]. Similar events have also been described in the case of several other oncogenes [16]. In particular, transformation of intestinal epithelial cells (IEC-18) by
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exogenous introduction of the oncogenic H-ras or v-src leads to the emission of bioactive exosome-like EVs containing the respective oncogenic cargo, including the genomic double-stranded H-ras sequences [19], which have also been described in other cellular contexts [20-23]. The presence of genomic DNA in a fraction of exosome-sized EVs is surprising, as such material is typically associated with cellular nuclei or apoptotic bodies, and it raises questions as to what is the nature of the material isolated as exosomal fraction from cancer cell supernatants through standard ultracentrifugation protocols. Here we show that ultracentrifugation is not sufficient to reliably isolate exosome-like EVs from certain cancer cell population. This is because ras- and src-mediated transformation enables substantial numbers of cells to enter and remain viable within the standard exosomal pellets obtained under vacuum and ultracentrifugation at 110,000 g. This was not observed in EV preparations of corresponding non-transformed cells or of less aggressive cellular populations, and it diminished in the presence of the oncogene inhibitor. This observation suggests a link between oncogenic transformation and the unusual resistance of cancer cells to extreme physical forces applied during ultracentrifugation. While the biological relevance of this property requires further study, we suggest that filtration is an essential step in EV preparation protocols involving highly transformed cancer cells. MATERIALS AND METHODS Cell culture and treatment conditions The following cell lines were used in the study: IEC-18 – non-tumourigenic, immortalized rat intestinal epithelial cell line; RAS-3 – tumourigenic, clonal IEC-18 subline that has been transfected with a V12 mutant, activated c-H-ras human oncogene; SRC-3 – tumourigenic, clonal variant of IEC-18 cells that has been transfected with the v-src oncogene; A431 – tumourigenic, human epidermoid carcinoma cell line harbouring oncogenic EGFR; NHA – nontumourigenic, normal human astrocyte cell line immortalized using human telomerase (hTERT), a gift from Dr. Guha, University of Toronto; U373P (or U373) – parental non-tumourigenic/indolent human glioblastoma cell line; U373vIII – tumourigenic variant of U373P cells, harbouring a constitutively active EGFRvIII oncogene [17]. IEC-18, RAS-3 and SRC-3 cells were grown as previously described [24]. A431, NHA and U373P cells were maintained in DMEM supplemented with 10% FBS (#080-150, Wisent Bioproducts) and 1% Penn/Strep (#15140, GIBCO). U373vIII cells were maintained under similar conditions with addition of 10% Tetracycline-free FBS (#081-150, Wisent Bioproducts). For experiments involving EV isolation, the media were supplemented with 10% EV-depleted FBS (ultracentrifugation at 150,000 g for 5 hours). Inhibition of SRC activity in SRC-3 cells was achieved by treating them with 20 µM PP2 compound (#P0042, Sigma) [25]. RAS-3-derived cell lines were developed by expanding the selected clones that survived
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ultracentrifugation, designated as PC1RAS-3, PC2RAS-3 and PC3RAS-3, and cultured as indicated. Isolation of extracellular vesicles EVs were obtained by ultracentrifugation either as previously described, or by a simplified protocol involving a slight modification in the number of centrifugation steps [17, 19]. Briefly, conditioned media were first centrifuged for 10 minutes at 400 g to remove cells and debris, followed by filtration through a 0.22 µm PES filter (#565-0020, Thermo Fisher Scientific). The filtrate was then ultracentrifuged for 1 hour at 110,000 g to isolate the pure EV fraction. In some cases, cells themselves were subjected to an extended ultracentrifugation, as indicated. Transmission electron microscopy (TEM) Cells and isolated EVs were washed once with 0.1M sodium cacodylate buffer, pH 7.4 and then fixed with 2.5% glutaraldehyde in the same buffer. The fixed samples were subjected to either ultramicrotomy for processing of monolayer cells, or whole mount negative staining for capturing of all EVs in the fixative. Tecnai 12 BioTwin 120 kV TEM was used to capture images. Clonogenic cell survival Following ultracentrifugation of the conditioned media at 110,000 g with or without filtration, the pellets were resuspended in complete media and cultured for 7 days prior to fixation with 10% formalin. Dishes were washed with distilled water and then stained for 20 minutes with 0.2% crystal violet in 20% methanol. After another wash with distilled water, images of clonal colonies were taken and their numbers quantified manually in the image of the entire well. MTS proliferation assay Viability or time-dependent increase in metabolic activity of growing cells was assessed using MTS assays (#G3580, Promega). Briefly, the indicated cells were plated in 96 well plates, grown for 1, 2 and 3 days, and processed according to the manufacturer’s protocol. To assess the recovery of viable cells following mechanical stress, 1,000 cells were ultracentrifuged directly at 110,000 g and MTS assay was performed at days 1, 2 and 3, as described. Data analysis Colony formation assays were performed at least twice with similar results. Experiments involving the same cell line across different studies (e.g. RAS-3) were done independently from one another. The statistical differences of resulting clonogenic recovery counts was analysed using computerised one-way ANOVA test, where comparisons were made to the reference cell line, as indicated. MTS proliferation assay results (N=4) were processed for statistical significance using the non-parametrical Mann-Whitney U test (Wilcoxon rank sum test). The threshold p-value of 0.05 was taken as a measure of significance.
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RESULTS AND DISCUSSION H-ras expressing cancer cells survive forces of ultracentrifugation In our prior studies on the cargo of EVs produced by cancer cells, we observed that this material contains bioactive oncogenes in a form of intact oncoproteins, mRNA and genomic DNA (gDNA) sequences [17-19]. This cargo can be recovered using either a complete EV preparation protocol involving 0.22 µm filtration of the culture supernatant, followed by ultracentrifugation at 110,000 g [19], or through a simplified (high yield) protocol containing pre-clearing and ultracentrifugation step alone (Fig. 1A). Thus, in the case of highly tumourigenic rat intestinal cancer cells harbouring the human mutant H-ras oncogene (RAS-3), transmission electron microscopy (TEM) reveals structural perturbations near the cellular plasma membrane and the presence of multiple exosome-like EVs, approximately 100 nm in diameter, in the ultracentrifugate of the cultured medium, with no evidence of debris or contaminating cells (Fig. 1B). However, the ample presence of the double-stranded gDNA in ultracentrifugates of RAS-3 conditioned media prompted us to examine whether, in spite of convincing TEM imagery, this material may contain rare viable RAS-3 cells that evade standard centrifugal pre-clearing (400 g). To do so, ultracentrifugation pellets of RAS-3 conditioned media were resuspended in medium and plated to test for the possible presence of tumour cell colonies (Fig. 1A). Interestingly, this assay revealed multiple colonies of clonogenic and proliferative cancer cells in spite of the extreme centrifugal force applied to them during the 1 hour ultracentrifugation in vacuum and at 110,000 g force (Fig. 1C). All such growth was eliminated when the media was passed through a 0.22 µm filter before ultracentrifugation, as per our standard protocol (Fig. 1AD), which continues to yield exosomal gDNA sequences [19]. These observations are important for at least two reasons. First, they enforce the notion that isolation of pure EVs produced by cancer cells should occur with inclusion of filtration steps to eliminate the rare cancer cells that may enter these preparations in spite of centrifugal pre-clearing. This is especially important in the case of bioassays aimed to demonstrate the EV-mediated transfer of biological properties from cancer to normal cells, as the presence of viable cancer cells could conceivably mimic such a transfer. Second, it is intriguing that at least some RAS-3 cells were able to survive extreme and prolonged (1 hour) forces of vacuum ultracentrifugation and retain their clonogenic potential under such harsh conditions. RAS-3 cells surviving ultracentrifugation exhibit sustained clonogenic potential To explore if survival under ultracentrifugation conditions represents a feature of some or all cells within the clonal RAS-3 population, we isolated individual colonies surviving this procedure and established three post-centrifugation (PC) clonal cellular sublines (PC variants – PC1RAS-3, PC2RAS-3 and PC3RAS-3).
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Fig. 1. Survival of H-ras-transformed cancer cells under conditions of vacuum ultracentrifugation. A – Schematic diagram illustrating EV preparations using the filtration (left) and no filtration (right) methods, as well as the workflow of the analysis presented. B – Vesiculation (arrows) observed at the surface of H-ras-transformed RAS-3 epithelial cancer cells. Transmission electron microscopy (TEM) images were taken at 23k (cells) and 49k (filtered EVs) magnifications, respectively. C – Unfiltered EV ultracentrifugate contains clonogenic RAS-3 cells. D – Filtered EV ultracentrifugate contains no clonogenic cells. E – The number of colonies recovered from the ultracentrifugates of conditioned
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media of RAS-3 and their variant cell lines (PC1RAS-3 – PC3RAS-3). These cells were expanded from clonal colonies grown previously from ultracentrifugates of RAS-3 conditioned media (p-values calculated using computerized one-way ANOVA versus RAS-3 cells). F – The viability or time-dependent increase in metabolic activity of RAS-3 and its variant cell lines were measured using MTS and found largely comparable except in PC1RAS-3 variants (N=4; *Wilcoxon rank sum test < 0.05). G – The viability or timedependent increase in metabolic activity of RAS-3 and its variant cell lines were measured after direct ultracentrifugation (1,000 cells; 110,000 g for 1 hour) by MTS and found largely comparable except in PC1RAS-3 variants (N=4; *Wilcoxon rank sum test < 0.05).
Equal number of these cells and their RAS-3 parental population were plated to generate conditioned media and this material was subjected to another round of ultracentrifugation and plating for clonogenic recovery of cancer cells. Notably, in the case of two out of three PC cell lines, the number of colonies recovered from such ultracentrifugate was higher, in one case significantly, compared to that of parental RAS-3 cells (Fig 1E). We also compared the viability of PC variants either by testing cells present in the culture conditioned medium (Fig. 1F) or when they were directly subjected to ultracentrifugation forces (Fig. 1G). These experiments, involving MTS assays, did not reveal consistent differences in the state or time dependent changes of metabolic activity (which may reflect growth or demise) between PC cell lines relative to their parental RAS-3 counterparts, even though one cell line (PC1RAS-3) had a somewhat higher metabolic rate and exhibited a more spindle morphology (data not shown). Pelleting trypsinized cells by vacuum ultracentrifugation at 110,000 g, once again, confirmed that both RAS-3 cells and their PC variants can withstand such conditions and retain their timedependent increase in metabolic activity, as measured by the MTS assay (Fig. 1G). This remained unchanged even after 3 hours of continuous ultracentrifugation (data not shown). Collectively, these observations suggest that RAS-3 cells are remarkably resistant to extreme centrifugal forces. Notably, the clonogenic recovery appears to differ between subpopulations of the clonal RAS-3 cell line, which may reveal the existence of cellular subsets (possibly new mutants) more resistant to this treatment. This raises the question as to whether such heterogeneity exists between other transformed and normal cells. Differential recovery of clonogenic cells from ultracentrifugates of transformed and non-transformed cell lines To explore whether the ability to withstand ultracentrifugation is specific to RAS-3 cells or common for cancer cells, we collected conditioned media from several human cell lines, including epidermoid and glioblastoma cells harbouring oncogenic EGFR (A431 and U373vIII cells), indolent but genetically transformed glioma cells (U373P), and immortalized but non-tumourigenic (normal) human astrocytes (NHA). Surprisingly, in contrast to RAS-3 cells, we were unable to consistently recover viable cells from centrifugates of any of these cell lines after their exposure to 110,000 g force, with a possible
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(paradoxical) exception of NHA cells, which, albeit inconsistently, generated individual colonies under such conditions (Table 1). It is of note that while A431 and U373vIII are tumourigenic, their relative in vivo aggressiveness is far less pronounced than that of RAS-3 cells, which give rise to a highly malignant and angiogenic tumours, and are resistant to anoikis and other insults [24, 26]. Therefore, it is possible that preferential survival of RAS-3 cells under extreme centrifugal forces is linked to some features of their malignant phenotype. Table 1. Clonogenic recovery of viable non-tumourigenic and tumourigenic cells from ultracentrifugates of culture conditioned media. Indicated cells with different tissue origin, aggressiveness and type of oncogenic transformation were cultured, conditioned medium collected and subjected to ultracentrifugation without filtration, as in the simplified protocol for EV preparation (Fig 1A). Pooled data from 6 independent experiments are shown, with numbers indicating colony counts from individual replicate wells. Of note, high level of transformation achieved by the expression of oncogenic H-ras or v-src rendered IEC-18 intestinal epithelial cells resistant to considerable centrifugal force. Nontransformed cells (IEC-18, NHA), indolent (U373P) and moderately transformed cancer cells (A431, U373vIII) exhibited a comparable, overall very low ability to yield viable cells in ultracentrifugates. P-values as indicated (one-way ANOVA), for comparisons with reference cell lines (* to ****, as indicated). Cells A431
Characteristics (origin and type of transformation)
NHA
Tumourigenic EGFR-driven carcinoma (human) Immortalized astrocytes (human)
U373P
Recovered P–value colony counts ANOVA (independent repeats) 3, 1 0.0014* 12, 1
0.0365*
Indolent glioma (human)
0, 1
Tumourigenic EGFRvIII-driven glioma (human) RAS-3 Highly aggressive H-ras-driven IEC-18derived carcinoma (rat) SRC-3 Highly aggressive v-src-driven IEC-18derived carcinoma (rat) SRC-3 + PP2 SRC-3 cells pre-treated with SRC inhibitor (PP2) IEC-18 Immortalized intestinal epithelium (rat)
0, 1 129, 82, 62, 157
0.0301* 0.3908**** 0.0301* 0.3907**** NA*
143, 96, 68, 121
0.9858*(NS)
20, 10
0.0196*** 0.0007** 0.0031*
U373vIII
10, 0, 3, 2
* - vs RAS-3; ** - vs IEC-18; *** - vs SRC-3, **** - vs NHA
Oncogenic transformation as a determinant of clonogenic cancer cell recovery upon ultracentrifugation RAS-3 cells are derivatives of the phenotypically normal, anoikis-sensitive and non-tumourigenic, but immortalized and clonogenic, rat intestinal epithelial cell line (IEC-18). From these cells, several highly aggressive tumourigenic variants were established following a single step transfection with H-ras or v-src
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oncogenes [24], as exemplified by RAS-3 and SRC-3 cells, respectively. Given the appreciable clonogenic recovery of viable cells from RAS-3 ultracentrifugates, it is reasonable to ask whether this property is cell line/typespecific or linked to a defined oncogenic event readily traceable in this simple model system. To address this question, IEC-18, RAS-3 and SRC-3 cells were used to generate conditioned media, which were subjected to 110,000 g ultracentrifugation and colony formation assays. Notably, the parental IEC-18 cells revealed negligible numbers of colonies following this procedure, while both RAS-3 and SRC-3 cells gave rise to ample clonogenic growth (Table 1). In this regard, the existence of specific and potent inhibitors of the SRC oncoprotein, such as PP2, offers an opportunity to examine whether the clonogenic survival of SRC-3 cells under centrifugal forces continues to depend on the transforming effect of the v-src oncogene. This appears to be the case, as conditioned medium of PP2-treated (but viable) SRC-3 cells generated reduced numbers of clonal colonies upon ultracentrifugation (Table 1). These results suggest that the ability to withstand centrifugal forces and contribute viable cells to the EV pellet is a function of oncogenic transformation, at least as it applies to cells exhibiting high expression of mutant H-ras or v-src oncogenes. In conclusion, our findings suggest that certain forms of oncogenic transformations confer cellular resistance to extreme physical forces. We serendipitously identified this influence while exploring protocols of ultracentrifugation involving centrifugal forces up to 110,000 g. This has several implications for techniques aimed at isolating EVs from cancer cell cultures (and biofluids) using simplified ultracentrifugation protocols. While such protocols may be effective in the case of normal cells and provide a high yield of EVs, our exploration of their use in the case of highly transformed cancer cells raises a cautionary note. The presence of viable and clonogenic cancer cells is not eliminated by the standard pre-clearing or extreme centrifugal forces, and may impact assays aimed at detecting various EV-specific molecular materials and influence the outcomes of relevant bioassays. These risks could be effectively addressed by maintaining a filtration step as an integral part of these experiments, one that effectively eliminates cellular contamination, but permits detection of EV-associated oncogenic cargo, including gDNA [19]. Unexpectedly, our study revealed a differential impact of the level and nature of oncogenic transformation on the survival of cancer cells under extreme physical challenges, as exemplified by prolonged vacuum ultracentrifugation. We observed that high levels of oncogenic H-ras and v-src expression, but not oncogenic EGFR, enable such survival, an observation that warrants further study as to how these respective oncogenes influence mechanosensory and physical properties of cancer cells. We and others have previously documented the resistance of RAS-3 and SRC-3 cells to detachment-induced cell death (anoikis), in contrast to their non-transformed IEC-18 counterparts [26]. This could be attributed to the relative uniqueness of signalling properties associated with the oncogenic RAS [27], and its impact on cytoskeletal organization [28-30].
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Of note is the fact that forces exerted on cells within the physical microenvironment are increasingly recognized as biologically important cues in cancer, including the cellular phenotype, cell shape, polarity and selection of stem cell populations [31-34]. The impact of the RAS pathway on mechanosensing and mechanical resistance is therefore of utmost interest. While forces applied to the cells at 110,000 g ultracentrifugation are artificial and likely exceed the conditions ordinarily encountered in vivo, cellular responses they elicit may signify the ability of cancer cells to survive relevant mechanical stresses, such as during the process of metastasis, invasive crossing of tissue barriers and expansion in confined tissue microenvironments, such as brain or bone. This includes behaviour of cells under conditions of movement, including rarely discussed circumstances such as aerial or space travel [35]. While few studies explored these questions in eukaryotic cells, certain bacteria have been documented to survive hyperaccelerative forces in excess of 400,000 g [36, 37], a finding that raises questions as to possible commonalities of these biological mechanisms and biomechanical principles to those operative in eukaryotic and diseased (cancer) cells. Implications of physical forces in human pathology remain to be thoroughly investigated. Acknowledgements. We thank our McGill colleagues J. Mui and L. Mongeon for their assistance in electron microscopy. This project was supported by a grant from the Canadian Institutes of Health Research MOP 11119 and 133424 (J.R.). J.R. is the Jack Cole Chair in Paediatric Haematology/Oncology. Support was also provided (S.C.) by MELS (Fonds de recherche du Quebec - Nature et technologies) and R. Tomlinson studentships. Infrastructure support was provided by the Fonds de la recherche en santé du Québec. REFERENCES 1. Lee, T.H., D’asti, E., Magnus, N., Al-Nedawi, K., Meehan, B. and Rak, J. Microvesicles as mediators of intercellular communication in cancer–the emerging science of cellular ‘debris’. Semin. Immunopathol. 33 (2011) 455-467. DOI: 10.1007/s00281-011-0250-3. 2. Thery, C. Exosomes: secreted vesicles and intercellular communications. F1000 Biol. Rep. 3 (2011) 15. DOI: 10.3410/B3-15. 3. Gyorgy, B., Szabo, T.G., Pasztoi, M., Pal, Z., Misjak, P., Aradi, B., Laszlo, V., Pallinger, E., Pap, E., Kittel, A., Nagy, G., Falus, A. and Buzas, E.I. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol. Life Sci. 68 (2011) 2667-2688. DOI: 10.1007/s00018011-0689-3. 4. Johnstone, R.M. Exosomes biological significance: A concise review. Blood Cells Mol. Dis. 36 (2006) 315-321. 5. Thery, C., Ostrowski, M. and Segura, E. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 9 (2009) 581-593. DOI: 10.1038/nri2567.
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