Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.
Copyright: © 2010 Buron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Competing interests: Authors ABS, NB and MP declare competing financial interests due to ownership interest in Mitologics SAS. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Apoptosis dysregulation has been shown to underly several pathologies including cancer [1], [2]. It is well established that diverse signalling events within apoptosis converge on mitochondria which undergo outer membrane permeabilization (OMP) triggering the release of soluble apoptogenic factors from the intermembrane space such as cytochrome c and a subsequent series of activation of a set of proteolytic enzymes, the caspases conducting to apoptotic dismantling of cell structure [3].
MOMP is under the control of members of the Bcl-2 protein family which includes (1) anti-apoptotic proteins like Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1/Bfl-1 containing all four Bcl-2 homology domains (BH1-4), (2) pro-apoptotic proteins like Bax, Bak, Bok lacking the BH4 domain and (3) pro-apoptotic BH3-only proteins like Bid, Bim, Bad, Bmf, Noxa and Puma [4]–[8]. In the direct activation model, induction of Bim or Bid is required for Bax or Bak to oligomerize and form pores in the outer mitochondrial membrane (MOM) [9], [10]. The anti-apoptotic proteins can block this process at the MOM by primarily sequestering Bax/Bak proteins [11]–[13]. In the indirect activation model [14], [15], BH3-only proteins can antagonize anti-apoptotic effect and liberate Bax/Bak proteins. It is still a matter of debate whether Bax and Bak may interact with proteins like VDAC (voltage dependent anion channel) and/or ANT (adenine nucleotide translocator) to regulate the permeability transition pore (PTP) [16]. At the mitochondrial level, the cytochrome c is distributed in two distinct pools: 15–20% in the intermembrane space and the larger fraction (80%) in the intracristae space [17]. Thus, BH3 mimetic peptide needs matrix remodeling to release the second pool of cytochrome c [18]. Other apoptotic factors like Omi/HtrA2 and Smac/DIABLO (caspase-dependent death effectors) or the apoptosis-inducing factor AIF and EndoG (caspase-independent death effectors) are released after MOMP.
The mitochondrial membrane permeabilization (MMP) process is often altered in cancer cells possibly as a result of PTP component overexpression [19], upregulation of anti-apoptotic members of the Bcl-2 family and/or downregulation of Bax [20]. These underly numerous anti-cancer strategies targeting components of the core cell death machinery to promote tumor cell death [21], [22]. These strategies are based on the use of BH3-mimicking peptides [14], [23], antisense [24] or RNA interference [25] against Bcl-2, and natural or synthetic small molecules which bind specifically to Bcl-2 family proteins. For instance screening approaches using nuclear magnetic resonance, structure-based design and combinatory chemical synthesis, led to the identification of ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-xL and Bcl-w but not Mcl-1 and A1/Bfl1 [26]. ABT-737 is considered to be a Bad-like BH3 mimetic because both ABT-737 and Bad BH3 peptide bind the same subset of Bcl-2 pro-survival proteins [27] and induce cytochrome c release in mitochondria obtained from “primed for death” tumor cells [28]. However, the weak affinity of ABT-737 for the pro-survival proteins Mcl-1 and A1/Bfl1 [26] might be a key determinant of tumor cell resistance to this compound [29].
We have set up a multiparametric screen on purified mitochondria to identify compounds inducing OMP of mitochondria isolated from cancer cell lines, but not of mitochondria isolated from non-cancerous cells. Among various compounds (from chemical, peptidic or proteic origins) described to target mitochondria, we found that only recombinant t-Bid, Bak BH3 and Bim BH3 peptides, and ABT-737 present a direct tumor-specific mitochondrio-toxicity and induce relatively large OMP due to Bax and Bak oligomerization. By further exploration of ABT-737-induced OMP at the cell-free mitochondrial level, we found that (1) cancer cell mitochondria from different sources differed in their sensitivity to ABT-737 correlating with different patterns of (outer) membrane-associated Bcl-2 family members and their interactions, (2) ABT-737 induces Bax, Bak, and Bim desequestration from Bcl-xL and Bcl-2, but not from Bcl-w or Mcl-1.
Mitochondria from both healthy tissue (mouse liver) and human tumor (PC-3, prostate) cell line were purified by isopycnic centrifugation in density gradients of Percoll. The isolated mitochondria were found highly intact as demonstrated by cytochrome c oxidase accessibility assay and flow cytometry FSC/SSC analysis [30]. Ultrastructural comparative studies of isolated mitochondria from liver or PC-3 tumor cell line reveal a relatively similar matrix/cristae organization despite a slight difference in density between tumor (low density) and liver (high density) mitochondria (Fig. 1A). Calcium (50 µM) induces an extensive outer membrane disruption in both healthy tissue and tumor cell line mitochondria followed by a swelling which is inhibited by cyclosporine A (CsA), indicating an intact and functional permeability transition pore in both mitochondrial types. Polarographic investigations were next carried out on liver and PC-3 mitochondria (Fig. 1B). Succinate oxidation was essentially dependent on ADP addition and a respiratory control index (RCI) of 3 associated with succinate oxidation indicated the functional integrity of mitochondria, including those isolated from tumor cultured cells. Similarly, mitochondria isolated from HT-29, HCT-116 and Jurkat cancer cell lines and HME-1 non-cancerous cell line presented high level of integrity and functionality (not shown).
A. Ultrastructural analysis of isolated mitochondria and their ability to swell. Electron micrographs were obtained after incubation of mitochondria isolated from healthy mouse liver, or PC-3 tumor cell lines untreated (NT) or treated with Ca2+ (50 µM) or with a 5 min-preincubation with cyclosporin A (CsA; 10 µM) or ruthenium red (RR; 1 µM) before calcium addition. The percentage of swollen mitochondria was <10% in the control and >80% 30 min after Ca2+ addition (n = 3). Scale bars 1 µm. B. Oxidative properties of isolated liver and PC-3 mitochondria. Traces represent oxygen consumption by isolated mitochondria (100 µg) after addition of the indicated reagents. Numbers along the trace are nmoles of O2 consumed per minute per milligram of protein. The respiratory control index (RCI) is calculated for each type of mitochondria as indicated in Materials and Methods. C. To evaluate mitochondrial swelling and ΔΨm loss, mitochondria isolated from healthy mice liver or PC-3 cell line were distributed in 96-well microplates and incubated for 30 min either with Ca2+ (100 µM) in presence (yellow) or absence (pink) of CsA (10 µM), with mClCCP (turquoise; 50 µM) or with t-Bid (purple; 1 nM). For quantitation of cytochrome c release, isolated mitochondria were treated with increasing concentrations of the t-Bid recombinant protein and mitochondrial supernatant was subjected to ELISA assays, given in percentage of release compared to 20 µg/ml alamethicin (Ala; 100% of cytochrome c release) (n = 3 independent experiments).
Isolated mitochondria were analyzed on a screening platform which allowed the quantification of the mitochondrial membrane permeabilization (swelling; Fig. 1C; left panel) plus mitochondrial transmembrane potential (ΔΨm; Fig. 1C, middle panel) using real-time spectrofluorimetry and cytochrome c release by ELISA as an index for MOMP (Fig. 1C; right panel). Real-time ΔΨm detection reflected inner membrane and respiratory chain alterations but did not permit to observe delayed ΔΨm in response to pro-apoptotic compounds. When incubated in hypotonic buffers, both normal and tumoral cell mitochondria did swell (loss of O.D. at 550 nm) in the presence of calcium in a CsA-dependent manner. However, the swelling amplitude was reduced in the case of tumor mitochondria in agreement with their lowest density compared to liver mitochondria. Calcium and mClCCP induced a rapid ΔΨm loss characterized by an increased fluorescence corresponding to Rhodamine-123 dequenching due to a decrease of the dye's concentration in depolarized mitochondria. We thus observed that the recombinant protein t-Bid had no effect on swelling and ΔΨm but induced cytochrome c release specifically in PC-3 (Fig. 1C), HT-29, HCT-116 and Jurkat (not shown) cell mitochondria in a concentration-dependent manner as indicated by ELISA analysis of the supernatants.
We next evaluated the effect of Bcl-2 inhibitors on mitochondria isolated from mouse liver, human non-cancerous (HME-1) and cancerous (PC-3) cells using 3 parameters: swelling, ΔΨm and cytochrome c release (Fig. 2A). The recombinant t-Bid protein induced cytochrome c release (without swelling and ΔΨm loss) from PC-3 mitochondria but had no effect on liver and HME-1 mitochondria at 100 nM. Some BH3 peptides (derived from Bak, Bim, Bax, Bad, Bid, Noxa and Puma) from human or mouse sources were also tested. Among these, only human Bak BH3 and Bim BH3 (Fig. 2A) induced mitochondrio-toxicity to tumor cell (PC-3) mitochondria, while being inactive at 100 µM on liver and HME-1 mitochondria. Noteworthy, even the corresponding mouse BH3 sequences are inactive on mouse liver mitochondria, excluding a misinterpretation due to species specificity (not shown). In contrast to the other small-molecule inhibitors evaluated in this study, only ABT-737 displayed tumor mitochondria specificity, inducing cytochrome c release from PC-3 mitochondria but not from liver and HME-1 mitochondria. The cytochrome c release from PC-3 mitochondria treated with t-Bid and ABT-737 occured without any swelling (Fig. 2A and electronic microscopy, not shown) or ΔΨm loss (Fig. 2A) during a 45 min-treatment, indicating that these conditions occurs a specific OMP. We then extended the study of ABT-737 effects to mitochondria isolated from the HCT-116, HT-29 and Jurkat cancer cell lines (Fig. 2B) and observed different sensitivity to ABT-737. Indeed, HT-29 mitochondria were much less sensitive to ABT-737 than PC-3, HCT-116 and Jurkat, these three laters presenting a similar level of sensitivity to ABT-737. These data suggested that ABT-737 induces cytochrome c release from various but not all mitochondria isolated from cancer cells.
A. Mitochondria isolated from mouse liver, human non-cancerous (HME-1) and cancerous (PC-3) cells were treated with increasing concentrations of t-Bid, Bak BH3, HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37, EM20-25 and ABT-737 before evaluation of mitochondrial swelling and ΔΨm loss. Alternatively, mitochondrial supernatant was subjected to ELISA assays for quantification of cytochrome c release. Effective concentration inducing 50% of the maximal effect (EC50) is given for swelling (100% of effect with 50 µM Ca2+), ΔΨm loss (100% of effect with 50 µM mClCCP) and cytochrome c release (100% of effect with 20 µg/ml alamethicin) (n = 3 independent experiments). B. Mitochondria isolated from mouse liver or HME-1, PC-3, HCT-116, HT-29 and Jurkat cell lines were incubated for 45 min at 30°C with increasing concentrations of ABT-737 and the supernatants were subjected to cytochrome c immunoblot (NT: untreated; Ala: alamethicin 20 µg/ml).
We subsequently investigated if ABT-737-induced OMP was selective to cytochrome c or might allow the release of other apoptogenic mitochondrial factors (Fig. 3). Isolated mouse liver, PC-3 and Jurkat mitochondria were treated with Bak BH3, ABT-737 or t-Bid and the supernatants subjected to immunoblotting. Smac/DIABLO (23 kDa) and Omi/HtrA2 (37 kDa) were released from PC-3 and Jurkat mitochondria whereas AIF (56 kDa) was not (Fig. 3), suggesting that these compounds induced a mitochondria remodeling not sufficient for AIF release. We next used isolated mitochondria from the Bax and/or Bak knock-out HCT-116 cell lines in which absence of Bax and/or Bak was checked by immunoblot (Fig. 4A). We found that ABT-737 induced cytochrome c release from Bax+/- and Bak-/- mitochondria but not from Bax-/- or Bax/Bak double knock-out mitochondria (Fig. 4B). This data pointed out the critical role of Bax in the mechanism of action of ABT-737. Furthermore, t-Bid and ABT-737-induced MOMP was controlled by an excess of Bcl-xL (Fig. 4C) or Bcl-2 (not shown) recombinant proteins, supporting the hypothesis of a formation of a specific channel at the outer membrane [31].
Isolated mitochondria from mouse liver, PC-3 and Jurkat cells were untreated (NT) or incubated either with alamethicin (Ala; 20 µg/ml; positive control), Bak BH3 peptide (10 µM), ABT-737 (1 µM) or recombinant t-Bid (1 nM) for 45 min. Mitochondrial supernatants were subjected to immunodetection of cytochrome c, Smac/DIABLO, Omi/Htra2 and AIF (Western blots are representative of 3 independent experiments). Note that cytochrome c (15 kDa), Smac/DIABLO (23 kDa), and Omi/Htra2 (37 kDa) but not AIF (56 kDa) are released from cancer cell mitochondria.
A. Total cell extracts from HCT-116 Bax +/-, Bax -/-, Bak -/- and Bax/Bak -/- (DKO) cell lines were subjected to Bax and Bak immunoblot to control their Bax and Bak content. B. Mitochondria isolated from HCT-116 Bax +/-, Bax -/-, Bak -/- and Bax/Bak -/- (DKO) cell lines were incubated with increasing concentrations of ABT-737 and the supernatant was subjected to immunoblot detection of cytochrome c (NT: untreated; Ala: alamethicin 20 µg/ml). C. Cytochrome c release induced by t-Bid and ABT-737 is inhibited by an excess of recombinant Bcl-xL. PC-3 mitochondria were incubated with ABT-737 (1 µM) or t-Bid (1 nM) for 45 min after a 5 min-pretreatment with recombinant Bcl-xL (100 to 400 nM) and the supernatant was subjected to anti-cytochrome c immunoblot (NT: untreated; Ala: alamethicin 20 µg/ml). Note that Bcl-xL strongly reduces both t-Bid and ABT-737-induced cytochrome c release (n = 2 independent experiments).
Having found that Bax remained bound to the mitochondrial OM even after a wash with an alkaline homogenization buffer (pH 11.6) (not shown) suggesting an insertion of Bax into the membrane [32], we further wanted to examine if ABT-737 might induce oligomerization of the Bax and Bak pools already associated to tumor cell mitochondria. Similar to t-Bid and Bim or Bak BH3 peptides, ABT-737, induced Bax and/or Bak oligomerization in PC-3 and Jurkat mitochondria, as objectived using the cross-linking agent 1,6-bismaleimidohexane (BMH; Fig. 5). Mutated [L78A; D83A] Bak BH3 peptide was inefficient to induce cytochrome c release and Bax/Bak oligomerization when added to PC-3 mitochondria (Fig. 5A). In PC-3 mitochondria which contain both Bax and Bak, a weak Bak oligomerization occured with BH3 peptides or ABT-737 suggesting a major role for Bax in triggering channels formation in this cell line (Fig. 5A; middle panels). We next used (±)-1-(3,6-dibromocarbazol-9-yl)-3-piperazin-1-yl-propan-2-ol identified by Bombrun and co-workers [33] as a Bax channel blocker (BCB) able to inhibit t-Bid-induced cytochrome c release [33], [34] (Fig. 5A). Pretreatment of cancer cell mitochondria with this BCB prevented cytochrome c release triggered by Bak BH3, Bim BH3, t-Bid or ABT-737 treatment (Fig. 5A). In addition, we found that BCB prevented Bax/Bak oligomerization in response to treatments with ABT-737, as well as t-Bid and Bak or Bim BH3 peptides (Fig. 5A and 5B).
Mitochondria isolated from PC-3 (A) and Jurkat (B) cell lines were incubated or not with the BCB, a Bax Channel Blocker (2 µM (±)-1-(3,6-dibromocarbazol-9-yl)-3-piperazin-1-yl-propan-2-ol) prior to treatment with 1 µM Bim BH3, 10 µM Bak BH3, 10 µM mutated Bak BH3, 1 nM t-Bid or indicated concentrations of ABT-737. Supernatants were analyzed for cytochrome c release (lower panels; NT: untreated; Ala: alamethicin 20 µg/ml) and mitochondrial pellets were treated with the irreversible crosslinker BMH (1 mM). Forty micrograms protein from each reaction was run on SDS-PAGE and immunoblotted with anti-Bax (A) or anti-Bak (A, B) antibodies. Bax/Bak oligomerization accompanies ABT-737-induced cytochrome c release which is inhibited by BCB.
Altogether, these data suggested that ABT-737 triggered the release of apoptogenic proteins from cancer cell mitochondria by formation of multimeric Bax/Bak channels as shown by correlation between Bax and Bak oligomerization and cytochrome c release (Fig. 5).
ABT-737-induced MOMP in cancer cell mitochondria is associated with particular complex disruptions, depending on the mitochondrial type
As differences in sensitivity were observed between the several mitochondrial-types used in this study, we analyzed the pro- and anti-apoptotic Bcl-2 family members associated to the mitochondrial membranes (Fig. 6). Among the anti-apoptotic proteins, Bcl-2 was only present in PC-3, Jurkat and HCT-116 mitochondria, while Bcl-w, Bcl-xL and A1 were detected in all mitochondrial types (Fig. 6A). Interestingly, Bcl-xL was quantitatively more important in cancer cell mitochondria than in their healthy counterpart. Anti-apoptotic Mcl-1L was present in large quantity in PC-3 and Jurkat mitochondria and in smaller quantity in HT-29 mitochondria. Concerning the pro-apoptotic proteins, while Bak was present in all mitochondrial types, Bax was present in PC-3, HT-29, HCT-116 and HME-1 mitochondria but not in Jurkat and liver mitochondria. Among the BH3-only activators, Bim was found in cancer cell mitochondria but not in those from HME-1 and liver (Fig. 6B) while Bid can not be detected in any of these mitochondrial types (not shown). Among the BH3 only sensitizers, Bad was detected at the PC-3, HT-29 and Jurkat mitochondrial membranes (Fig. 6B), while Puma, Noxa, Hrk, Bik, Bok and Bmf were not (not shown). Bcl-2, Bcl-xL and BH3 only sensitizers (ex Bim) might well to be key actors even if it is difficult from such proteomic analysis to explain the differences in sensitivity to ABT-737. Indeed it is noteworthy that HME-1 mitochondria have neither Bim, nor Bcl-2 and only low level of Bcl-xL, which might distinguish them from sensitive cancer cell mitochondria.
Total cell extracts (TE) and mitochondrial extracts (M) from PC-3, HT-29, Jurkat and HCT-116 cancer cell lines or from healthy HME-1 cell line and mouse liver were analyzed by Western blot for detection of the anti-apoptotic (A) Bcl-2, Bcl-xL, Bcl-w, Mcl-1L and A1 proteins and the pro-apoptotic (B) Bak, Bax, Bim, Bad and Mcl-1S proteins.
As ABT-737 is acting by complex disruption between pro- and anti-apoptotic proteins, we next investigated some complex disruptions by co-immunoprecipitation in PC-3, HT-29 and Jurkat mitochondria treated with ABT-737 (Fig. 7). Whatever the cell line we detected similar bindings: Bcl-xL to Bax and Bak, Bcl-2 to Bax and weakly to Bak, Mcl-1 only to Bak (Fig. 7) and Bcl-w to Bax (not shown). We observed that ABT-737-induced cytochrome c release is correlated with Bax, Bak (Fig. 7) and Bim (not shown) liberation from Bcl-xL and Bcl-2. However, ABT-737 had no effect on Bak and Bim sequestration by Mcl-1 (Fig. 7), or Bax sequestration by Bcl-w (not shown), these complexes remaining after treatment. These results suggested that Bax, Bak and Bim liberation from Bcl-2 and Bcl-xL in response to ABT-737 was responsible for channels formation and cytochrome c release in PC-3 (Fig. 8) and Jurkat mitochondria. In contrast, HT-29 mitochondria containing less Bim and being deprived of Bcl-2 were less sensitive to ABT-737 treatment, suggesting a major role for Bcl-2 and Bim in ABT-737 sensitivity.
Mitochondria isolated from PC-3 (A), HT-29 (B) and Jurkat (C) cells were untreated (NT) or treated with t-Bid (Bid; 2 nM) or ABT-737 (ABT; 1 µM) before to be immunoprecipitated by the antibodies directed against the Bcl-2, Bcl-xL and Mcl-1 anti-apoptotic proteins. Mitochondrial total extracts (TE; positive control of immunoblot; 25 µg) were used as control while a mitochondrial lysate was subjected to immunoprecipitation process without antibody (C; negative control of immunoprecipitation). Thus Western blot analysis was performed to determine bindings between anti-apoptotic proteins and pro-apoptotic Bax and Bak proteins (representative Western Blots of 3 independent experiments).
A. Bax, Bak and Bim are sequestred by Bcl-2, Bcl-xL and Mcl-1 at the outer mitochondrial membrane. B. In response to ABT-737, Bax, Bak and Bim proteins are liberated from Bcl-2 and Bcl-xL but not from Mcl-1L. Thus Bim can directly enhance Bax and Bak oligomerization triggering MOMP formation (C) and release of pro-apoptotic proteins such as cytochrome c in the cytosol.
In this study, we used high quality controlled isolated mitochondria to compare the effects of putative Bcl-2 inhibitors and try to explore the mechanism of action of ABT-737. We used five different parameters to evaluate their integrity and functionnality: cytochrome c oxidase accessibility to exogenous cytochrome c (not shown), respiratory control values, capacity for matrix swelling, transmembrane potential values and release of apoptogenic factors like cytochrome c (OMP) (Fig. 1). Comparision of compounds-effect on each mitochondrial type requires similarly high levels of purity and intactness of mitochondrial preparations whatever their sources (cultured cells or healthy tissue). This was solved by large-scale cell cultures and purification of mitochondria by differential centrifugations plus Percoll density gradient. Using this method, both isolated mouse liver and cancer cell mitochondria present similar quality and response to calcium (Fig. 1).
Surprisingly enough most compounds identified as Bcl-2 inhibitors were found to act on healthy mitochondria at least on one integrity parameter. For instance, we observed that HA-14.1, Chelerythrine, Gossypol and EM20-25 induced MMP in mouse liver mitochondria, while other Bcl-2 family inhibitors were found to be inactive (YC-137 and TW-37). Appart from t-Bid, Bak BH3, Bim BH3 which are from proteic origins, only ABT-737 demonstrated selective tumor mitochondrio-targeting indicated by OMP and release of pro-apoptotic factors (Fig. 2). Previous observations have proven that ABT-737 can induce OMP either when mitochondria originate from cells “primed” by death signals (for instance in IL-3-deprived lymphocytes [28], or in TNF-pulsed HeLa cells [35]), or when isolated mitochondria are co-treated with BH3 peptide (for instance with Noxa BH3 on MEF mitochondria [13]). For the first time, we demonstrated that ABT-737 can itself induce OMP on mitochondria isolated from unprimed tumor cell lines. Concerning t-Bid, our isolated liver and HME-1 healthy mitochondria were not sensitive to the recombinant protein t-Bid. This absence of effect on liver mitochondria could be explained by the high purity and stability of our mitochondrial preparations. Bcl-2 family proteins detected on both normal and cancer cells mitochondria (Fig. 6) remind present after alcaline washes (not shown) indicating that they are not associated by electrostatic interaction with the mitochondrial membranes and are not coming from residual cytosol or endoplasmic reticulum.
The recombinant t-Bid protein, Bak BH3, Bim BH3 and ABT-737 triggered a release of apoptogenic proteins from PC-3 and Jurkat mitochondria by formation of channels large enough to release proteins such as Omi/HtrA2 (37 kDa) (Fig. 3). OMP appears independent on PTP since it is not inhibited by known PTP inhibitors like ADP, cyclosporin A and bongkrekic acid (not shown). The absence of mitochondrial membrane alterations (no swelling and ΔΨm loss) (Fig. 2A) and the release of the smallest apoptotic factors under treatment (Fig. 3) suggested that ABT-737 induced the formation of a specific channel and not a mitochondrial membrane rupture, similarly to the Bax[53–86] BH3 peptide in Polster et al. [36]. Accordingly, discriminative release of apoptogenic factors has already been shown in isolated HeLa mitochondria treated with t-Bid [37]. This finding is compatible with the previous description of an apoptosome-dependent loop where downstream caspases need to be activated to trigger mitochondrial release of AIF and EndoG, secondary to the release of cytochrome c, Omi/HtrA2 and Smac/DIABLO [37]. In cellular model, ΔΨm loss and cytochrome c release were simultaneously detected in response to ABT-737 [38], [39] contrary to what was observed with our conditions in cell-free system. Our screening method seems to be a real time process that allows detection of direct and early effects of compounds on mitochondria, without interferences induced by cytosolic compartment.
We have also shown that (1) HCT-116 Bak-/-, but not Bax-/-, mitochondria are sensitive to ABT-737 (Fig. 4A), (2) ABT-737-induced cytochrome c release on PC-3 mitochondria are controlled by an excess of Bcl-xL (Fig. 4C) and (3) inhibition of Bax and Bak oligomerization by BCB is sufficient to block cytochrome c release (Fig. 5A and B). These findings indicate that equilibrium between pro-apoptotic and anti-apoptotic members of the Bcl-2 family plays an essential role in the ABT-737 mechanism of action.
We have thus demonstrated that Bax and Bak oligomerization at the PC-3 mitochondrial membrane is induced by Bak and Bim BH3 peptides, t-Bid or ABT-737 treatments (Fig. 5A), Bax and Bak both being inserted as a monomeric form in untreated normal (HME-1) and tumoral (PC-3) cell mitochondria. However, numerous studies have been performed showing Bax oligomerization and subsequent membrane insertion using recombinant Bax and isolated mitochondria or liposomes [40]–[42]. These studies have led to opposite conclusions on the kinetic of Bax pores activation. However, more recently, it has been shown that oligomerization of Bax occurs at the mitochondrial level rather than in the cytosol [43]–[45]. Thus, using c-myc null cells, Annis and co-workers showed that Bax-induced mitochondrial permeabilization results from oligomerization of transmembrane monomers rather than insertion as preformed oligomers [43].
Some Bcl-2 family proteins, such as the BH3 only activator Bim or the anti-apoptotic proteins Bcl-2 and Mcl-1L are especially present at cancer cell mitochondria. In contrast with previous observations [28], [29], [46], Mcl-1L expression at the mitochondria was not sufficient in our hands to prevent MOMP formation in response to ABT-737. For instance, PC-3 and Jurkat mitochondria are sensitive to low concentrations of ABT-737 despite a high Mcl-1L content (Figs 2 and 6), while HT-29 mitochondria with low level of Mcl-1L are relatively resistant to ABT-737. We show here that at the molecular level, ABT-737 allows pro-apoptotic proteins Bcl-2 and Bcl-xL but neither Mcl-1L nor Bcl-w to liberate Bax, Bak and Bim (Figs 7 and 8). Bim, as activator of Bax and Bak oligomers, plays a key role in ABT-737-induced apoptosis [47]. This suggests that sensitivity to ABT-737 depends on Bim presence and on the balance between the quantity of Bcl-2 and Bcl-xL versus Mcl-1L and Bcl-w, explaining resistance of some mitochondrial types, deprived of Bcl-2 (HT-29 mitochondria) or both Bcl-2 and Bim (HME-1 and liver mitochondria). Interestingly, HME-1 mitochondria are less sensitive to t-Bid than cancer cell mitochondria despite the presence of Bax and Bak (Fig. 6). This observation suggests a slight difference in Bax and Bak regulation in healthy and cancer mitochondria isolated from cultured cell lines. Extended investigations are needed to explain this difference.
Finally, the comparative approach based on isolated “pathological” versus “healthy” mitochondria appears to be a usefull tool to identify Bcl-2 inhibitors and investigate their mechanism of action on a particular cell type. It also represents a reliable, fast, and predictive screening tool, tailored for selecting series or compounds with selective toxicity profile against mitochondria from cancer cell lines and devoid of toxicity against healthy mitochondria.
Liver mitochondria were isolated from 6 weeks old BALB/cByf female mice (Charles River, Saint Germain sur L'arbresle, France) as previously described [30]. Animal housing, care and application of experimental procedures were conducted in compliance with the European Community guidelines for the care and use of experimental animals (Animal Health Regulations, Council Directive No. 86/609/EEC of 24th November 1986). The experimental procedure on mice was rewieved and approved by the Bichat - Debré Hospitals Ethics Committee. Purified organelles were re-suspended in homogeneization buffer (300 mM sucrose, 5 mM TES pH 7.2, 0.2 mM EGTA, 1 mg/ml BSA). Mitochondria were also isolated from human mammary gland epithelial cells immortalized by stable expression of the human telomerase reverse transcriptase [48] (hTERT-HME-1; ATCC) and human cancer cell lines (PC-3, prostate adenocarcinoma (NCI); HT-29, colon adenocarcinoma (ATCC); Jurkat, acute T cell leukemia (ATCC); HCT-116, colon adenocarcinoma, deficient or not for Bax and/or Bak (from Dr. Peter Daniel and Prof. Bert Vogelstein). Briefly, adherent cells were harvested with Trypsin/EDTA, centrifuged at 750 rpm for 10 min, washed in buffer A (100 mM sucrose, 1 mM EGTA, 20 mM MOPS, pH 7.4 and 1 mg/ml BSA) before cell break with a Dounce homogenizer. The suspension was centrifuged twice at 2 500 g for 5 min and the resulting supernatant at 10 000 g for 10 min at 4°C. The pellet was resuspended in buffer B (300 mM sucrose, 1 mM EGTA, 20 mM MOPS pH 7.4, 1 mg/ml BSA and 1 mM PMSF) and the homogenate was layered on a two phase percoll density gradient. After centrifugation, mitochondria (layered at the interface) were removed, washed with homogeneization buffer for 10 min. at 10 000 g, and resuspended in homogeneization buffer. To ensure quality of mitochondrial preparations, samples were subjected to various assays for integrity and functionnality including cytochrome oxydase accessibility, respirometry and FSC/SSC FACScan (BD Bioscience, Germany) analysis in the presence or absence of Mitotracker™ green (ΔΨm insensitive) and Mitotracker™ red (ΔΨm sensitive) as described in [30].
Our screening platform is dedicated to the real-time co-monitoring of mitochondrial swelling and ΔΨm. Freshly isolated mitochondria are distributed in 96-well plates in buffer D (200 mM sucrose, 5 mM succinate, 10 mM MOPS pH 7.4, 1 mM H3PO4, 2 µM rotenone and 10 µM EGTA) supplemented with 1 µM rhodamine 123 (Rh123; Molecular Probes™, Invitrogen, Cergy Pontoise, France) followed by the addition of serial dilutions of small compounds or synthetic peptides. Absorbance at 545 nm and Rh123 fluorescence (excitation 485 nm, emission 535 nm) are recorded during 30 cycles of 1 min using a fluorescence multi-well plate reader (Infinite, Tecan®, Männedorf, Switzerland). CaCl2 (50 µM) and mCICCP (20 µM) treatments were considered as the 100% baseline for the swelling and ΔΨm loss, respectively. The EC50 are the concentrations corresponding to 50% of maximal swelling and 50% of maximal ΔΨm loss at 30 min.
Isolated mitochondria (20 µg proteins) were incubated with 20 µg/ml Alamethicin (Ala, positive control, 100% baseline for ELISA), small molecules or synthetic compounds in buffer D for 30 or 45 min at 30°C. After a 7 min centrifugation at 10 000 g, proteins contained in supernatant were analyzed for quantification of cytochrome c release using ELISA kits from MBL (Cliniscience, Montrouge, France) for liver mitochondria and from Biosources (Invitrogen) for tumor cell lines mitochondria and/or runned on NuPAGE® 4–12% Bis-Tris gels (Invitrogen) and transfered to nitrocellulose using the iBlot™ Dry Blotting System (Invitrogen). Subsequently the membrane was blocked for 1 h with 5% low fat milk in TBS-0.1% tween-20 (TBST) and incubated with anti-cytochrome c mouse monoclonal IgG2b antibody (BD Bioscience Pharmingen; 1/500); or anti-Smac/DIABLO (Calbiochem; 1/3000), anti-Omi/HtrA2 (R&D Systems, Lille, France; 1/2000), anti-AIF (Upstate Biotechnology, Millipore, Billerica, MA;1/1000) rabbit polyclonal IgG antibodies. After 1 h-washes with TBST, the membrane was treated with horseradish peroxidase-coupled anti-mouse IgG (Promega, Charbonnière, France; 1/2500) or anti-rabbit IgG (Amersham Biosciences, Piscataway, NJ; 1/3000) for ECL detection (Amersham Biosciences).
A 20 mM stock of 1,6-bismaleimidohexane (BMH; Pierce) prepared in DMSO was added to treated mitochondria at a 1∶20 dilution in buffer D. After 1 hour at 30°C, mitochondria were centrifuged 10 min at 12 000 g and dissolved in 4x NuPAGE sample buffer (Invitrogen) with DTT (dithiothreitol) before SDS-PAGE electrophoresis.
Isolated mitochondria (600 µg) were incubated or not with t-Bid (2 nM) or ABT-737 (1 µM) during 40 min at 30°C. After centrifugation for 10 min at 10 000 g, mitochondria were lysed by incubation at 4°C during 15 min in 1 ml of CHAPS buffer (Hepes 50 mM, KCl 150 mM, EDTA 1 mM, EGTA 1 mM, CHAPS 0.2%, NP40 0.05%, NaF 5 mM and PMSF 1 mM). After centrifugation for 10 min at 10 000 g, supernatant was incubated for 16 h at 4°C with protein G agarose (Pierce) and antibodies: mouse anti-Bcl-2 (Santa Cruz clone C-2), anti-Mcl-1 (Santa Cruz clone 22) or rabbit anti-Bcl-xL (Cell Signaling). The precipitate was washed 4 times with CHAPS buffer and boiled 5 min in loading buffer before immunoblot analysis.
Isolated mitochondria were fixed with 2% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.2 for 3 h at 4°C. After 2 washes with 0.2 M sucrose in 0.1 M Na-cacodylate buffer, pH 7.2, the specimens were then postfixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate, dehydrated in gradual ethanol (30–100%) and embedded in Epon. 70 nm thin sections were collected onto 200 mesh cupper grids, counterstained with uranyl acetate and lead citrate before examination with a Zeiss EM 902 transmission electron microscope at 80 Kvolt (MIMA2, Electron Microscopy Platform-GPL, Jouy-en-Josas). Microphotographies were acquired using MegaView III CCD camera and analysed with ITEM software (Eloïse SARL, Roissy, France).
Isolated mitochondria were incubated in a magnetically stirred 1.5 ml cell with a Clark type oxygen electrode (Hansatech Instruments Ltd, Norfolk, UK) thermostated at 37°C, in 500 µl of a medium consisting of 0.3 M mannitol, 10 mM phosphate buffer (pH 7.3), 10 mM KCl, 5 mM MgCl2 and 1 mg/ml BSA as previously described [49]. ADP addition causes a sudden burst of oxygen uptake when the ADP is converted into ATP characterized by an actively respiring state (state 3) respiration followed by a slower rate after all the ADP has been phosphorylated to form ATP (state 4). The ratio [state 3 rate] / [state 4 rate] which is called the respiratory control index (RCI) indicates the tightness of the oxidative phosphorylation, thus referring to the respiratory chain functionality and the quality of the mitochondrial preparation. The inhibitor Oligomycin A which blocks respiration in coupled mitochondria and the uncoupling agent mClCCP were added in the case of tumoral cell mitochondria.
Conceived and designed the experiments: NB EJ ABS. Performed the experiments: NB MP MB DD CR ML CP. Analyzed the data: NB MP ABS. Contributed reagents/materials/analysis tools: PR ABS. Wrote the paper: NB PR EJ ABS.
8. Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9: 47–59.
11. Willis SN, Chen L, Dewson G, Wei A, Naik E, et al. (2005) Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 19: 1294–1305.
We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it.
Copyright: © 2015 Ayuso et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Funding: This work has been supported by National Research Program of Spain (DPI2011-28262-c04-01) and by the project "MICROANGIOTHECAN" (CIBER-BBN, IMIBIC and SEOM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Chemotaxis is the process by which cells migrate along a concentration gradient towards a chemoattractant. Chemotaxis plays a critical role in many pathologies, including inflammation [1–3] and autoimmune diseases [4] as well as cancer [4–7], and also many developmental and tissue remodeling processes, including embryogenesis and wound healing [8, 9]. Therefore, techniques that enable detailed scrutiny of the chemotactic process are important tools for drug discovery as well as basic biology.
A number of different protocols are used to study cell migration and chemotaxis. The Boyden chamber assay [10] has been one of the most popular assays to study chemotactic response for many years. Although this method is versatile, it has some limitations; for example, it does not allow direct observation of cells during migration processes. A number of other techniques have been developed to monitor cell movement in chemotactic experiments, including the under-agarose gel [11], agarose spot [12], Zigmond chamber [13], Dunn chamber [14], and Insall chamber [15] assays. Although each of these assays has its own merits, they generally do not allow gradient monitoring over time, and in addition, gradient control and reproducibility can be challenging. Many of these shortcomings can be overcome by using microfluidic systems that have come to the fore as powerful tools to study chemotaxis [16–18]. A large variety of microdevices have been designed to generate chemotactic gradients, and one of the most well-known is the Premixer Gradient Generator[19]. These microdevices rely on microchannels that split and recombine two different liquids several times in order to generate the desired gradient. Other common designs to generate molecular gradients are based on the use of microjets. In such microdevices molecules are transported through small microchannels by convection to the culture area. This culture area is much larger than the microchannels, causing the transported molecules to lose momentum. Therefore, within the culture area, diffusion dominates over convection, leading to gradient generation[20]. The microdevice presented herein belongs to another type of gradient generators based on porous hydrogel membranes. Hydrogels possess a porous structure which opposes high resistance to liquid flow. However, molecules can diffuse passively through these pores, enabling the gradient generation. Microdevices designed with a central chamber to accommodate a hydrogel, and flanked by lateral microchannels to perfuse different media, have proved to be robust tools to generate gradients and study cell responses [21–26].
Whilst observations of single cell migration with the existing techniques are very useful, they are in some way distanced from more realistic cell migration scenarios, in which cells are part of a multicellular system. Many reports have shown that solid tumor cells can migrate and invade by different mechanisms than individual cells [27, 28]. For example, gliomas are examples of isolated invading tumors, whereas epithelial tumors seem to invade by collective movements [27, 29]. With these differences in mind, it is important to consider that to date; none of the previously described assay protocols allow the study of this collective cancer cell migration in multicellular spheroids in response to chemotactic gradients, which leaves a major gap in our understanding of the process. Here we present a microfluidic device of simple design that permits real time observation of multicellular spheroids embedded in a hydrogel. Moreover, it allows the study of their chemotactic behavior under conditions generated by introduction of a chemoattractant gradient through lateral microchannels. We directly compare the migration of multicellular spheroids embedded in collagen in a microfluidic system to those in a well-plate. We will show, that in response to chemotactic gradient, epithelial squamous cancer cells behave differently when cultured as isolated cells than as multicellular spheroids. This is the first example of such an investigation utilizing a microfluidic device.
In order to easily accommodate multicellular spheroids, microdevices were designed with 400 μm diameter inlets and with 400μm wide and 300μm high microchannels. The central microchamber is 1000μm wide and 300μm high. Microdevices were fabricated using SU–8 photolithography combined with an SU–8 to SU–8 bonding process[30]. This fabrication process was inspired by previously reported work [31–33] and is described in detail in the supporting information (S1 File). Briefly, different SU–8 layers were patterned with the desired geometry in order to create the bottom, middle and top layers. The central microchamber is separated from lateral microchannels by a series of pillars, which allow hydrogel confinement due to the hydrogel’s surface tension. When it is injected within the microdevice, the hydrogel interface gets pinned between these pillars, filling only the central microchamber without entering the lateral microchannels. After hydrogel polymerization, different media can be injected through lateral microchannels with no mixing except for passive diffusion, which allows the establishment of diffusion-controlled gradients.
Human oral squamous cancer cell line OSC–19 [34] was a generous gift from Dr Faye Johnson, MD Anderson Cancer Center, Houston, USA. Human glioblastoma cell line U–87 MG was purchased from American Type Culture Collection (ATCC; LGC Standards, Middlesex, UK). Prior to use, both cell lines were maintained as monolayers in RPMI 1640 growth media (R7388) supplemented with 10% (v/v) fetal bovine serum (FBS)(F1051), 1 mM sodium pyruvate (S8636) and 2 mM L-glutamine (G7513) (All from Sigma–Aldrich, Dorset, UK). For the remainder of the manuscript, RPMI 1640 growth media supplemented with FBS is referred to as “complete” whereas RPMI 1640 growth media not supplemented with FBS is referred to as “incomplete”. When required, cells were trypsinized and used as a suspension in growth media.
Fluorescein diacetate (FDA) (Sigma F7378), Rhodamine B (Sigma, R6626) propidium iodide (PI) (Sigma P4170), Cascade Blue®-10 kDa and Tetramethylrhodamine (TRITC)-70 kDa dextrans (Life Technologies, D1976 and D1818) were used as solutions in phosphate-buffered saline (PBS) (Lonza BE17-516F). PBS 5X was prepared by dissolving of 1 pack of PBS powder (Sigma P3813) in 200 ml deionised water. For immunofluorescence primary anti-Ki–67 antibody (abcam ab92742), secondary donkey anti-rabbit alexa-546-conjugated (life technologies A10040) and DAPI (life technologies D1306) were used.
OSC–19 and U–87 MG multicellular spheroids were generated using the hanging drop method [35] with methylcellulose [36]. Briefly, 12 g of high viscosity methylcellulose (Sigma, M0512) were dissolved in one liter of RPMI1640, and centrifuged at 4000 g for 2.5 hours. Only the clear supernatant was used in the next step. Cell suspension (105 cells/ml) and this methylcellulose solution were mixed in a 4:1 v/v ratio, and 25 μl droplets (2x103 cells) were placed on the lid of Petri dishes. Sterile water was placed on the bottom of the dishes to prevent evaporation from droplets, the lids were replaced and the Petri dishes incubated at 37°C and 5% CO2. After 1 day, a single well-defined spheroid was generated per drop. To ensure the response of the spheroids only depends on exposure to the external chemotactic gradient of FBS, traces of serum were removed from the spheroids prior to the experiments by washing with incomplete RPMI. To do this, each spheroid was transferred to a single well from a flat bottom 96 well-plate with 200 μl of incomplete media and allowed to stand for 30 minutes. The mean spheroid diameters were 248±18 μm for OSC–19 and 236±5 μm for U–87 MG.
15.4 μl of collagen type I (4.88 mg/ml, Corning 354236), 0.37 μl of aqueous NaOH (1N, Sigma 655104) and 4 μl of PBS 5X were mixed and added to a suspension of fifty spheroids in 30 μl of incomplete media. The final concentration of the collagen in this stock mixture was 1.5 mg/ml. The spheroid suspension in collagen was injected into the microdevice, and 5 μl of spheroid suspension was placed on top of the inlet to prevent hydrogel evaporation during polymerization. The microfluidic device was then placed into an incubator (37°C, 5% CO2) for 15 minutes to allow collagen polymerization. For embedding of individual cells, a similar procedure to the one described above was used to obtain a hydrogel mixture; in this case a cell suspension of 2x103 cells/μl was mixed with the collagen mixture and injected into the microdevice.
FDA (5 μg/ml), Rhodamine B (1 μg/ml), PI (4 μg/ml), 10 kDa dextran (10 μM) or 70 kDa dextran (10 μM) were pipetted into one of the lateral microchannels whilst PBS was perfused through the other one. To visualize gradient evolution, time-lapse confocal microscopy images were taken.
Chemotaxis experiments were performed within the SU–8 based microdevices (Fig 1A). Complete growth media was perfused through one lateral microchannel, whereas incomplete growth media was perfused through the opposite side (Fig 1B). Microdevices were observed on the microscope with temperature and CO2 control systems, with conditions set at 37°C and 5% CO2. Brightfield images were taken every 5 minutes.
(A) SU–8 fabricated microdevice. (B) Experimental scheme. (C) OSC–19 spheroid of 2000 cells in the hanging drop. (D) Same spheroid after embedding in 1.5 mg/ml collagen hydrogel within the microdevice. Treatment of the spheroids with FDA/PI after 48 hours in a hydrogel in a 96-well plate or within the microdevice shows the spheroid is intensely green (E and F respectively) whereas less than five red cells where observed (G and H respectively), Z projection of the whole spheroid is shown. Scale bar is 200 μm.
Samples were fixed for 30 min with 4% paraformaldehyde (VWR J61899-AP), permeabilized using 0.1% Triton-X–100 (Sigma T8787) and blocked with 5% BSA (Sigma A2058) in PBS. Samples were then incubated overnight with primary antibody (1/50 in 2.5% BSA and 0.05% Triton X–100), and secondary antibody (1/200) was used under the same conditions. DAPI staining was performed overnight and after a washing step samples were visualized.
To assess cell viability and visualize the gradient, confocal images were taken using a Nikon Eclipse Ti microscope equipped with a C1 modular confocal microscope system. Images were collected at different focal planes (300 μm in the “z” direction with 10 μm steps) for each microdevice and well-plate control. In order to analyze cell viability a “Z projection” was performed using the Fiji® (http://fiji.sc/Fiji) maximum intensity projection algorithm. Isolated cell migration was tracked and quantified using the Fiji® manual tracking plugin, whereas OSC–19 spheroid migration was quantified by measuring manually the spheroid-occupied area at different times. This spheroid-occupied area was drawn to show the collective invasion under the different chemotactic conditions.
Experiments were repeated at least three times. OSC–19 invasion results are presented as mean ± standard error. Statistical analysis was performed using SPSS software, and statistical significance was set at p < 0.05. The normal distribution was tested by the Kolmogorov-Smirnov test, and the Student´s t-test was used for the data analysis.
We compared the viability of cells within the spheroids inside the microfluidic device to those in a well-plate plate 24 hours after implantation by treating both with FDA/PI solutions to detect viable and dead cells. We observed that spheroids within the microdevice remained intact during injection within the microdevice. Cell viability was measured in spheroids located within the microdevice as well as in those placed in the well-plate. Results showed more than 95% of the cells were viable in both conditions (Fig 1) and no necrotic core was observed.
We next set out to gauge the exposure of the spheroids and individual cells within the microdevice to a chemical gradient. To assess how this exposure is time dependent, Rhodamine B (1 μg/ml) was added to one side of the confined collagen hydrogel in the microdevice and allowed to diffuse across the hydrogel. Time-lapse confocal images showed the gradient evolved along the full width of the central microchamber (Fig 2A and 2B).
(A) A complete image of the microchamber 5 minutes after red fluorescent Rhodamine B was perfused through the right lateral microchannel. (B) Evolution of fluorescence intensity of Rhodamine B along the whole microchamber width (as highlighted in yellow in A) over time. (C) An image of an OSC–19 spheroid after 5 minutes of FDA perfusion through the right lateral microchannel. (D) Fluorescence intensity along the section of the spheroid (as highlighted in yellow in B) after 5 minutes of FDA perfusion. (E) Fluorescence intensity within the spheroid after 1 hour of FDA perfusion. (F) Fluorescence intensity along the spheroid perimeter after 1 hour of FDA perfusion through only one lateral microchannel (gradient condition-blue line) or through the hydrogel (control condition-red line). Images corresponding with the spheroid middle focal plane are shown. Scale bar is 100 μm.
We next set out to evaluate the spheroid gradient sensing ability using FDA that was added to one side of the collagen hydrogel within the microdevice and allowed to diffuse across the hydrogel. In live cells, FDA enters through the cell membrane and once inside the cell, it is transformed by the action of esterases into the non-permeable green-fluorescent compound Fluorescein [37]. Thus, cells within the spheroid became fluorescent once they were exposed to the FDA perfused through one of the lateral microchannels. As can be seen (Fig 2C), cells on the surface of the spheroid did indeed show fluorescence, however, the intensity of this fluorescence was not uniform across the spheroid boundary at any stage during the 1 hour experiment (Fig 2D and S2 File). Instead, those cells on the spheroid surface closer to the diffusing chemical front show more fluorescence intensity, suggesting that they are exposed to more FDA. Over the length of the experiment, fluorescence intensity gradually builds up not only in the front of the spheroid, but also to the sides and the back of the spheroid (Fig 2E). Interestingly, whilst there is significant infiltration of the chemical into the spheroid, FDA appears to reach the back of the spheroid before reaching the core. Fluorescence intensity measurement along the spheroid perimeter showed a maximum centered on the region closest to the microchannel perfused with FDA, and decreasing as the distance increases (Fig 2F, red line). This pattern is wholly consistent with a chemical front moving across the microchamber and washing over the spheroid rather than penetrating through it. Since we have already shown the whole of the spheroid contains viable cells (see above) we conclude that this is not due to a necrotic core, but an inability of FDA to penetrate through the spheroid at a rate comparable to the one it diffuses through the medium.
When FDA was flushed manually through the hydrogel, exposure of the spheroid to the chemical was uniform and fluorescence intensity along the spheroid perimeter showed homogeneous distribution (Fig 2F, blue line).
Here a FDA/Rhodamine B gradient has been characterized. However in the FBS there is a complex mixture of different growth factors. These different growth factors have different molecular weights, which have a deep impact on the diffusion velocity. To illustrate this, the diffusion profile of fluorescent 10kDa and 70kDa dextrans was recorded using confocal microscopy (Fig 3). The results showed how the larger dextran took a longer time to diffuse through the hydrogel. Many of these FBS growth factors have a molecular weight within this 10 kDa–70 kDa range [38], therefore these results provide a good model for the time-scale of the diffusion profile and show the influence of molecule size on gradient evolution.
(A) Cascade blue–10 kDa and TRITC–70 kDa dextran at 10 μM were injected in the left microchannel and confocal images are shown after 4 hours. Fluorescent 10 kDa and 40 kDa diffusion profile is compared at 5 min (B), 60 min (C) and 4 hours (D) post-injection.
We next turned our attention to the invasion of OSC–19 spheroids. It is already a well-established principle that cancer cells directionally invade towards a serum enriched environment [17, 18]. To demonstrate that OSC–19 spheroids invade in response to serum, we first carried out a migration experiment outside the confines of the microdevice. OSC -19 Spheroids embedded in collagen were seeded in a 96 well-plate and cultured in complete medium. In this experiment, spheroids experience a uniform (non-gradient) concentration of FBS. We observed that the rate of spheroid invasion depended on the concentration of FBS. Whilst no invasion was observed in 0% FBS, and little invasion in 1% FBS, in 10% FBS there was significant, but non directional and quite random, migration (Fig 4).
OSC–19 spheroids were embedded in collagen hydrogel in a six well plate and growth media supplemented with 0% FBS (A and B), 1% FBS (C and D) and 10% FBS (E and F), was applied over them. Photos were taken at embedding time (0 hours) and after 20 hours. Scale bar is 200 μm.
In the microdevice, a FBS gradient was achieved by injecting one of the lateral microchannels with complete media and the other microchannel with incomplete media, establishing a gradient of FBS across the central microchamber. In a control experiment, both lateral microchannels are charged with complete media. For OSC–19 spheroids inside the device, when there was no gradient of serum across the microdevice, i.e. when both channels are charged with the complete growth media, we observed no directional invasion (Fig 5). When the OSC–19 spheroids were subjected to a FBS concentration gradient we observed a clear collective invasion of cells towards that concentration gradient (Fig 6). This migration has also been captured in a time-lapse movie (S1 Movie).
OSC–19 spheroids were embedded in collagen hydrogel within the central microchamber. Media supplemented with 10%FBS was perfused through both lateral microchannels. Spheroid invasion is shown after (A) 15 hours, (B) 23 hours, (C) and 30 hours. (D) Area occupied by the spheroid at different times is represented in the graph. Scale bar is 100 μm.
OSC–19 spheroids were embedded in collagen hydrogel within the central microchamber. Media supplemented with 10%FBS was perfused through right hand lateral microchannel, whereas incomplete media was used in the other. Spheroid invasion is shown after (A) 15 hours, (B) 23 hours, (C) and 30 hours. (D) Area occupied by the spheroid at different times is represented in the graph. (E) Comparison between invaded area under gradient and non-gradient conditions, asterisk denotes difference between both halves is statistically significant (p-value < 0.05). Scale bar is 100 μm.
Whilst this invasion was clearly directional with movement towards the FBS gradient, it was not uniform. Indeed, we consistently observed cell offshoots with significantly more invasive response. This is entirely consistent with the proposal that in collective migration, leader cells open up the path of invasion and other cells follow them [39]. It is interesting to note that in view of our earlier observation which showed that chemoattractant waves engulf the spheroid rather than go through it, we believe the invasion is mostly initiated from the outer layers of the spheroid.
Significantly, this invasion is both aggressive (vide supra) and coordinated, resulting in cells invading collectively.
Isolated OSC–19 cells where embedded in a collagen hydrogel within the microdevice, and the response to a 10% FBS gradient was evaluated. Isolated cell trajectories were tracked, showing no preferential migratory direction and very low net displacement (Fig 7A and 7B, S2 Movie, at least ten cells were analyzed). Cell viability was measured using FDA/PI at the end of the study, revealing a majority of cells to be viable (Fig 7C). Interestingly, analysis of time-lapse images revealed that spheroid cell velocity and directionality were much higher compared to the isolated cells (Fig 6 and S1 Movie). This observation is in excellent agreement with previous work showing how epithelial cells migrate more intensely when they are allowed to migrate by a collective movement [40, 41]. Given the relatively short time scale of the experiment and the relatively long distance the cell front moves during the experiment, it is unlikely that proliferation plays a major role in the invasion process. Nevertheless, in order to measure any contribution from proliferation to the invasion process, we checked for the expression of Ki–67, a well-established cellular marker of proliferation, in OSC–19 as well as U–87 MG spheroids, by immunofluorescence. For OSC–19 spheroids, the results showed that cells located behind the leading edge of invading cells were negative (S3 File) for Ki–67 although as expected, the population of cells at the invading edges where positive for Ki–67. Whilst the cells on the leading edge are expected to be more proliferative, this result demonstrated that proliferation as well as migration contributes to the invasion process.
(A) Individual OSC–19 cells were embedded in collagen hydrogel within the central microchamber. Media supplemented with 10%FBS was perfused through one lateral microchannel, whereas basal media was used in the other. Tracks of migrating cells are shown. (B) Individual cell trajectories are plotted, showing those with a net displacement to the right in red, and those displaced to the left in black. (C) Isolated OSC–19 cell viability after 30 hours under gradient conditions, viable cells are shown in green. Scale bar is 100 μm.
We also investigated migration of U-87MG spheroids. Since these cells are non-endothelial, we expected the pattern of migration to be different. Again, to demonstrate that U87-MG cells migrate in response to serum, we first carried out a migration experiment outside the confines of the microdevice. U87-MG spheroids embedded in collagen were seeded in a 96 well-plate and covered in complete growth medium to experience a uniform (non-gradient) concentration of FBS. As before, we observed that the rate of spheroid migration depended on the concentration of FBS, however in this case, expansion of the spheroid was significantly faster and even at 0% FBS, we observed some expansion of the spheroid (Fig 8). To account for the greater responsiveness of U87-MG cells to FBS, a less steep concentration gradient of FBS was applied across the microchamber by injecting one of the microchannels with growth media containing 1% FBS and the other microchannel with incomplete media (containing 0% FBS). We found that the less steep concentration gradient helped in visualizing migration patterns as there were fewer cells (Fig 9 and S3 Movie). When proliferation was measured, no Ki–67 positive cells were observed (S3 File), demonstrating that U–87 invasion was due only to the migration process.
U87-MG spheroids were embedded in collagen hydrogel in a six well plate and growth media supplemented with 0%FBS (A and B), 1% FBS (C and D) and 10% FBS (E and F), was applied over them. Photos were taken at embedding time (0 hours) and after 20 hours. Scale bar is 200 μm.
(A) Spheroid was embedded in collagen hydrogel within the central microchamber. Media supplemented with 1%FBS was perfused through one lateral microchannel whereas basal media was used in the other. Brightfield image with overlaid tracks of migrating cells after 12 hours. (B) Individual cell trajectories are plotted, showing those with a net displacement to the right in red, and those displaced to the left in black. Scale bar is 100 μm.
Chemotaxis is the principle mechanism by which cells migrate within multicellular organisms. Within this general description, chemotactic processes play different and quite significant roles in many biological processes, and in particular in many developmental and pathophysiological events. Therefore, in vitro models that enable the study of chemotaxis have become important tools in biology, particularly those models that provide a closer analogy with physiological systems. In this context, the microdevice we describe herein, and its application to the investigation of the migratory aptitude of multicellular spheroids is a particularly powerful tool.
It is already well established that multicellular spheroids provide a number of advantages over 2D cultures [42–44]. They are considered a more complete model that takes into account cell-cell interactions akin to that observed in real tumors. Our investigations demonstrate further advantages for using microdevices in the study of multicellular spheroids. The control of the chemotactic gradient across the microchamber, coupled to the ability to observe and closely monitor the system over time, makes this a powerful technique for the study of the chemotactic process. In recent years, collective invasion has been proposed as the dominant migration mode during epithelial tumor development [27], and the protocol outlined here enables that process to be observed and analyzed in vitro.
Furthermore, microfluidic devices such as the one designed and used here, can clearly have a much wider application. Work is ongoing in our groups to expand the scope of this technique, to study the migratory aptitude towards chemotactic cytokines (chemokines) which are known to play a significant role in cancer cell migration and metastasis; and to study co-cultured multicellular spheroids containing mixed population of cells which better represent the tumor microenvironment.
34. Yokoi TY, Yamaguchi A, Odajima T, Furukawa K. Establishment and characterization of a human cell line derived from a squamous cell carcinoma of the tongue. Tumour Research. 1988;23:43–57.