Dr Laura Fourmois

Dr Florent Poyer

Aude Sourdon

Delphine Naud-Martin

Sounderya Nagarajan

Dr Rahima Chennoufi

Dr Eric Deprez

Marie-Paule Teulade-Fichou
email: mp.teulade-fichou@curie.fr

Florence Mahuteau-Betzer

Dr D A Sourdon

Dr S Naud-Martin

Dr M.-P Nagarajan

Dr F Teulade-Fichou

Mahuteau-Betzer

Modulation of cellular fate of vinyl triarylamines through structural fine tuning: to stay or not to stay in the mitochondria?

Keywords: Fluorescent probes, Chemical Biology, Organelle imaging probes, Mitochondria

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Introduction

Fluorescent probes are powerful tools for cellular imaging. In the last decades, there has been a strong interest in the design of organelle-targeted fluorescent probes as well as fluorophores that are responsive to cellular changes. [1] Among the numerous chemical probes developed, those targeting mitochondria are particularly attractive thanks to the central role of this organelle in the regulation of the cellular death. Indeed, mitochondria is not only the power unit of the cell but is also involved in apoptosis [2] and in diverse cellular pathways such as intracellular calcium signaling and reactive oxygen species production. As dysregulation of apoptosis can lead to several disorders such as cancer, [3] neurodegenerative, [4] autoimmune or ischemic diseases, [5] the status of mitochondria can be used as a diagnosis tool. In this context, a number of probes have been designed to monitor the production of ROS, [6] the mitochondrial pH changes [7] or the mitochondria membrane potential variation (m). [8] .The latter, in particular, is a key parameter of the mitochondrial function and is characterized by a highly negative value m =-180mv), which results from the charge imbalance across the inner mitochondrial membrane generated by the oxidative phosphorylation chain. Since membrane permeabilization and m are intrinsically connected and related to apoptosis,m can be used as indicator of apoptosis and necrosis. [9] Therefore the design of m responsive probes is of great interest for imaging cellular death as well as for diagnosis and chemotherapeutic intervention. Most of the mitochondria-targeted fluorescent probes [10] are lipophilic cations characterized by a delocalized charge (DLCs) whose uptake is driven by the electrostatic force developed by the negative gradient across the inner mitochondrial membrane. [11] As a result the concentration of DLCs can be up to 100 -1000 times higher in the mitochondrial matrix than in the cytosol. [12] Nonetheless owing to the very active metabolism of ions exchanges due to the electron transport chain, the trapped DLCs may move back and forth across the mitochondrial membranes depending on (transient) variation of the membrane potential that in turn is directly influenced by the mitochondria health status. For instance, conventional fluorescent cationic stains such as Rhodamines (e.g. Rhodamine 123 and analogues), are readily sequestered into mitochondria and are released to the cytosol when m dissipates. Therefore, this class of dyes cannot stain mitochondria in fixed (dead) cells since the fixation treatments induce massive mitochondrial membrane depolarization. Conversely, MitoTracker TM probes (a family comprised of rosamine and cyanine derivatives) are able to stain mitochondria in both fixed and live cells. [13] This particular behaviour results from the presence of one or several chlorobenzyl groups on the dye aromatic skeletons which induce covalent reaction with the thiol functions of proteins in mitochondria making the relocalisation of MitoTracker probes impossible and therefore independent of variation of m. On the other hand, variation in m can be signalled by specific probes although only a handful of those are available. The most popular is the carbocyanine dye JC-1, which [14] accumulates in the mitochondrial matrix where it forms red-emissive aggregates whereas upon membrane depolarization its concentration decreases lead to the formation of green-emitting monomers. Thus monitoring of m by JC-1 relies on the ratiometric emission between the monomers and the aggregates. However, although JC-1 is widely used, its uptake, concentration in the extracellular medium and incubation time need to be carefully controlled in order to get a clear response depending on its aggregation or disaggregation status. [8] To circumvent this drawback and to provide efficient imaging tools for mitochondria status monitoring, recent reports proposed new probes based on alternative strategies such as the relocalization from mitochondria to other organelles under mitochondria membrane depolarization. [15] In this context, we undertook to explore further the properties of TPA dyes, a series that we pioneered several years ago and that possess remarkable fluorescence characteristics for cellular bioimaging. [16] TPA dyes, bearing two or three cationic branches were initially designed for nonlinear (two-photon) absorption and were shown to display high photo-stability, while being live-cellpermeant. Furthermore these dyes are fluorogenic upon binding to the DNA minor groove, which switches on their fluorescence with dramatic quantum yield enhancement. [16b] Accordingly, in fixed cells TPA-induce bright and stable nuclear DNA staining with excellent contrast whereas in live cells their behaviour is radically different, [17] as they remain sequestered in mitochondria. Most interestingly upon light excitation either visible (488 nm, 1photon absorption) or NIR (760-860 nm, 2-photon absorption), [18] TPA leave the mitochondria and rapidly translocate to the nucleus. This phenomenon is concomitant with ROS production and apoptosis. Therefore, on one hand TPA rank amongst the brightest DNA markers developed so far in fixed cells [16b] and on the other hand, act as photoactivatable mitochondria-targeted proapoptotic agents in live cells. [17][18][19] However, the structural determinants influencing the mitochondrial sequestration as well as the capacity to translocate out of the mitochondria in absence of light activation and in particular the dependence with regard to the mitochondrial membrane potential have not been examined in detail. In addition, the question of whether TPA mitochondrial localisation is in part driven by mtDNA binding remained unanswered. This question in particular is of broad interest as several studies have reported that cationic dyes could target secondary DNA structures in mtDNA but without providing direct evidence. [20] Furthermore finding new labels with a variety of excitation wavelengths for visualizing mtDNA is also of great interest as mentioned in a recent report. [21] Therefore, stimulated by the recent impetus for dye targeting mitochondria, it was decided to address these questions through molecular engineering of the TP-2Bzim compound (Figure 1), which represents the best candidate of the TPA series in terms of brightness and DNA binding affinity. Hence in the present work, we report on structural modifications of the reference compound TP-2Bzim devoted to modulate the lipophilic /cationic charge balance, which led to four new Bzim derivatives. Firstly, we modified the number of charges through the synthesis of the neutral analogue TP n -2Bzim and the monocationic analogue TP 1+ -2Bzim then the number of vinyl branches was reduced affording the monobranched compound TP 1+ -1Bzim (Figure 1). Finally, the triphenylamine core has been replaced by its trinaphtylamine counterpart providing the more lipophilic analogue TN-2Bzim. This core modification was previously shown to strongly improve the two-photon absorption properties due to the higher electron-richness of the TNA core. [22] Next we studied the photophysical properties and the cellular imaging of this new panel of dyes in order to determine the physico-chemical parameters responsible of their cellular behaviour. In the whole, the systematic comparison of the dyes in fixed and live cells indicated that subtle structural changes strongly affect the partition between mitochondria and nucleus, the translocation dynamics and presumably the initial mitochondrial membrane localisation.

Results and Discussion

Design and synthesis TP-1Bzim was prepared following the same procedure reported for the synthesis of TP n -2Bzim and TP-2Bzim: [16b] a Wittig reaction between the mono-formyl triphenylamine and the (Nmethyl benzimidazole)-methyltriphenylphosphonium chloride afforded compound 1 in 47% yield, subsequent methylation allowed the formation of TP-1Bzim in quantitative yield. The monocationic compound TP 1+ -2Bzim has been synthesized in two steps as follows: a mono Wittig reaction was performed on the commercially available precursor 4,4'-diformyltriphenylamine to afford the monosubstituted -aldehyde 2 in high yield (84%). This intermediate compound was then converted into TP 1+ -2Bzim by a Knoevenagel reaction using 1,2,3trimethylbenzimidazolium iodide (see Scheme 1). The synthesis of TN-2Bzim was also achieved in two-steps as follows: the commercially available 2-naphtylamine and 6-bromo-2naphtaldehyde was coupled via a Buchwald-Hartwig amination to give the 6,6' diformyltrinaphtylamine 3 in a moderate yield (38%). This step involves two coupling reactions: the first one between 2-naphtylamine and 6-bromo-2-naphtaldehyde and the second one between the resulting dinaphtylamine intermediate of lower reactivity and a second equivalent of 6-bromo-2naphtaldehyde. A Knoevenagel reaction between dialdehyde 3 and 1,2,3-trimethylbenzimidazolium iodide afforded TN-2Bzim (see Scheme 1) in a moderate yield (31%). 2,3-trimethyl-1Hbenzo[d]imidazole-3-ium iodide, piperidine, EtOH, 80°C, 18h, 38% ; (e) Pd(OAc)2, P(t-Bu)3, Cs2CO3, toluene, reflux, 48h, 38%; (f) 1,2,3-trimethyl-1Hbenzo [d]imidazole-3-ium iodide, piperidine, EtOH, 80°C, 15h, 31%.

Photophysical properties

The optical properties of TP-1Bzim, TP n -2Bzim, TP 1+ -2Bzim, TN-2Bzim were characterized in glycerol and in buffer (pH 7.2) with or without DNA at room temperature (Table 1). The properties of the TP-2Bzim are provided here for comparison. All derivatives show a maximal absorption between 411 and 450 nm in aqueous buffer and strong molar absorption coefficients between 21 200 to 42 500 M -1 .cm -1 . The four cationic compounds are poorly fluorescent in aqueous buffer with quantum yield ranging from 0.006 down to < 0.001, in consistency with our previous reports. Conversely the neutral derivative TP n -2Bzim exhibits substantially higher emission (5fold that of the monocationic TP 1+ -2Bzim). This illustrates the polarity effect of water towards highly polar excited states (push pull) and is consistent with the lower polarity of the neutral compound. Since this compound series is known to be sensitive to the environment viscosity, the optical properties were also assessed in glycerol (see Figure S1). In all cases, the dye fluorescence is strongly enhanced in glycerol with a maximum emission between 520 to 637 nm and with quantum yields from 0.08 to 0.58 (Table 1). As shown previously, the enhancement of fluorescence in glycerol is attributed to reduced rotational motions of the aromatic rings around the vinylic bond inducing inhibition of the non-radiative de-excitation pathway. As expected, the addition of charges, from TP n -2Bzim to TP-2Bzim, increases the transfer of charges between the donor core (TP/TN) and the terminal acceptor Bzim groups, leading to a bathochromic shift of the emission (from 561 nm to 620 nm in water, from 520 nm to 602 nm in glycerol). The extension of the conjugation for TN-2Bzim as compared to TP-2Bzim also leads to an expected bathochromic shift in the absorption wavelength but also in the emission wavelength (from 620 to 638 nm in buffer and from 585 to 614 nm in glycerol).

Since TP-2Bzim is a potent DNA-interactive compound that binds in the minor groove of AT-rich duplex DNA with submicromolar affinity, [15b] it was of interest to evaluate the impact of the structural modifications on the dye /DNA interaction. To this end UV and fluorimetric titrations were performed using a 14-mer palindromic sequence (5 ′ -CGCGAAATTTCGCG, Drew AT) forming a short duplex mimicking AT-rich B-DNA. For all compounds, absorption is strongly decreased upon addition of DrewAT with a red-shift of the maxima for TP 1+ -2Bzim and TN-2Bzim indicating a decrease in polarity of the dye environment arising from interaction with DNA (Table 1, Figure 2). Worth noting is the presence of an isosbestic point in the case of TP 1+ -2Bzim (500 nm, Figure 2A) similarly to that observed for TP-2Bzim (Figure 2B) indicating the presence of two species in equilibrium whereas none is observed for the neutral, the monobranched and the trinaphtyl derivatives (Figure 2A). On the opposite, the variation in emission strongly differs from one compound to another: TP 1+ -2Bzim retains the fluorescence turn-on property of TP-2Bzim and displays a remarkable 45-fold increase in quantum yield at saturation in DrewAT (Figures 2A andB). As well in both cases the emission is blue-shifted as has been observed for certain DNA dyes, [23] and is attributed to changes in the dipole moment upon excitation. The monobranched monocationic analogue TP-1Bzim shows a significantly lower fluorescence enhancement (4.5) denoting the importance of the 2 Bzim branches moiety for the fluorescence increase. In contrast the fluorescence of TP n -2Bzim is strongly quenched upon adding DrewAT (Figure 2A)  [c]  F [d] F×   (abs)

TP-1Bzim

Buffer [e] 31 ; DrewAT: 10 mol equiv of DrewAT. [b] λabs: maximum absorption wavelength (nm); λem: maximum emission wavelength (nm). [c] ε: molar absorption coefficient (mol -1 •L•cm -1 ). [d] ΦF: fluorescence quantum yields were measured using rhodamine 101 in ethanol as reference. [e] 2PA cross section (GM). [f] data from ref. [16b] whilst the fluorescence emission of TN-2Bzim is poorly affected (2-fold increase) (Figure 2A). The titration curves resulting from the fluorescence variations (Figure S2, supporting information) allowed to determine 1:1 binding for TP 1+ -2Bzim and TP-1Bzim with Kd values in the submicromolar range showing a strong association although 100-fold lower than that of the parent biscationic TP-2Bzim [15b] as expected. For the neutral and TNA derivative the weak variations observed reveal a poor association, between the dyes and the DNA which is likely nonspecific and could correspond to external binding or partial insertion in grooves. In particular, in the case of TN-2Bzim although it harbors the same biscationic charge as TP-2Bzim, it is likely that the 3D geometry of the trinaphtyl core is too bulky and therefore not appropriate to fit in the minor-groove contrary to what was shown for TP-2Bzim. [15b] This indicates that although the electrostatic forces contribute significantly to DNA binding, other weak forces resulting from the tight fit of the two vinyl branches of TP-2Bzim and TP 1+ -2Bzim into the AT-rich minor groove seems to be a strong determinant of the interaction.

Finally since high two-photon cross-section have been reported for neutral trinaphtylamines (TNA) [22] , the two-photon absorption spectra of TN-2Bzim was measured in DNA by the TPEF (twophoton excitation fluorescence) method (see Figure S3, supporting information). The 2PA spectrum shows a very high cross-section (= 2500 GM at 830 nm), which is two-fold higher than that of TP-2Bzim (= 1080 GM at 840 nm) thus confirming the advantage of the TN core for optimizing two-photon absorption properties. [22] However, due to its lower fluorescence quantum yield and its low binding to DNA, TN-2Bzim displays a lower two-photon brightness in presence of DNA (×F = 250) than its TP counterpart (×F = 583). For the same reason the one-photon brightness of this compound is also significantly impacted (Table 1).

Cellular localization

Next, we investigated the cellular localization of the TPA-TNA dyes and before their biocompatibility was evaluated in Hela cells via CellTiter Glo assay (SI, Table S1). The probes show no significant effect on cell viability at 2 µM after 24h of incubation and can be used for live-cell imaging. Then the cellular localization of the four probes was examined both in fixed and live HeLa cells and again compared to that of TP-2Bzim which is known to display nuclear localization in fixed cells and mitochondrial localization in live cells. Interestingly TP 1+ -2Bzim was the only one to exhibit a behaviour similar to that of TP-2Bzim but with a significantly lower brightness as directly visualized by the comparison of figures 3E,F and figures 3G,H. This correlates well with the weaker DNA affinity and with its photophysical properties determined in solution as the brightness of TP 1+ -2Bzim in drewAT is 4.5-fold lower than that of TP-2Bzim (F×=5363 and 23976 respectively) (Table 1). As well excellent contrast is obtained with the two dyes in consistency with their DNA light-up properties (Figure 2, Table 1).

On the opposite, the three other dyes (TP-1Bzim, TP n -2Bzim TN-2Bzim) did not cause nuclear staining in fixed cells (Figure 3A,C,I) but rather showed a diffuse cytoplasmic staining, thereby confirming either their poor association to DNA neither their low fluorescence enhancement factor, which is not sufficient to cause nuclear labelling in cells. The same cytoplasmic labelling is observed in live cells, but with a spot-like pattern for the two cationic compounds TP-1Bzim and TN-2Bzim (Figure 3B,J) suggesting mitochondrial localization, whereas the signal remains diffuse for the neutral compound TP n -2Bzim (Figure 3D). We previously demonstrated that TP-2Bzim is mainly localized in the mitochondria using different organelle trackers in live cells. [16][17] Following a similar approach, co-localization experiments were performed with Mitotracker® Deep Red (MTDR) to confirm the localization of TP-1Bzim, TP 1+ -2Bzim and TN-2Bzim (Figure 4). Excitation at 633 nm led to the near infra-red fluorescence of MTDR (650-800 nm, colored in red) while excitation at 488 nm led to a yellow-red fluorescence typical of the TPA probes (colored in green to allow overlay). The overlay of the pseudo colored images results in a strong yellow color (Figure 4A,B,D) thereby confirming the mitochondrial localization in all cases similarly to what was observed for the parent compound TP-2Bzim (Figure 4C). Spatial co-localization was analyzed using the Van Steensel's method (see Figure S4) and the intensity of co-localization was confirmed by calculating Pearson's correlation coefficient (≈0.80 for, TP-2Bzim and TN-2Bzim). The monocationic TP-1Bzim and TP 1+ -2Bzim showed a Pearson's correlation coefficient with a slightly lower value (0.69-0.7) which might result from difference in emission intensities between the probes and the commercial dye. Taken together these experiments point out that the probes and commercial mitochondrial tracker present similar spatial distribution evidencing mitochondrial sequestration. Finally, the high two-photon cross-section of TN-2Bzim prompted us to reproduce cellular imaging under two-photon excitation which revealed a bright mitochondrial staining under low laser power (see Figure S5, SI). As suggested in a number of studies, the mitochondria trapping of dyes can be dependent on the cell line and in particular preferential accumulation in mitochondria of cancer cells has been reported (justified by the higher m in cancer cells vs normal cells). Thus, the cellular localization of TP 1+ -2Bzim, TP-2Bzim and TN-2Bzim was assessed in human primary fibroblasts CCD19-Lu (see SI Figure S6-7). The distribution patterns obtained are identical to those observed in Hela cells with the same labelled compartments. Thus, at least from this two-cell lines comparison, it can be inferred that the intracellular distribution of the dyes is not significantly affected by the status of the cell line.

Labelling of mitochondrial DNA

UV and fluorescence titrations have shown that TP-2Bzim and TP 1+ -2Bzim interact strongly with DNA whilst the TN-2Bzim interacts weakly. As the three compounds stain mitochondria in living cells, we presumed that they could also bind and label mitochondrial DNA (mtDNA) at least for TP-2Bzim and TP 1+ -2Bzim. Currently the classical approach to assess mtDNA binding is to carry out colocalization experiments with Picogreen, a commercial mtDNA probe. In live cells Picogreen is able to label both nucleus and mitochondria subcompartment in which it induces dot-like staining (i.e. foci) typical of mtDNA binding. However, this colocalization analysis could not be performed in the present case due to large overlap between the emission spectra of Picogreen and the Bzim dyes. Hence, we adopted an indirect strategy based on the use of 2',3'-dideoxydicitidine (ddC), a well-known inhibitor of mtDNA replication. [24] Treatment by ddC reduces the amount of mitochondrial DNA, causing a strong decrease in mitochondrial staining intensity. HeLa cells were incubated with ddC for 4 days and then incubated 24 h with Picogreen or with the Bzim probes. Cells without ddC treatment were used as control for comparison (see Figure 5). The cells treated with ddC did not show any changes in membrane integrity or morphology in comparison to non-treated control cells. As expected Picogreen® induces solely nuclear staining in ddC treated cells whereas nuclear and mtDNA labelling are observed in the control non-treated cells (Figures 5A,E), which is clearly indicative of effective blocking of mtDNA replication. Conversely when using the three probes (TP 1+ -2Bzim, TP-2Bzim and TN-2Bzim) the mitochondrial labelling patterns were not significantly modified by the ddC treatment (compare Figure 5B,C,D and Figure 5F,G,H). These results suggest that the Bzim compounds are not able to reach the mtDNA. In order to confirm this behaviour, we handled experiments with a mitochondrial DNA-lacking cell line (i.e. HeLa rho 0 cells) that represents another useful tool to explore interaction with mtDNA (see Figure S8). [25] Picogreen was used as a positive control and MTDR that anchors covalently in inner membrane proteins was used as a negative control . Both HeLa and HeLa rho 0 cells were incubated with TP 1+ -2Bzim and TP-2Bzim, the best DNA ligands of the series. No changes in the staining patterns were observed with the two dyes, indicating that mitochondria labelling is not affected by the absence of mtDNA (Figure S8B,C,F,G) which is also the case of the negative control MTDR (Figure S8D,H). By contrast Picogreen labeled only the nucleus in cells depleted in mtDNA (Fig. S8 E). The results of these two experiments constitute indirect but strong evidence that the mitochondrial staining induced by Bzim dyes is not resulting from interaction with mtDNA suggesting that the dyes are not able to reach the matrix where multiple copies of mt DNA are located. Presumably interactions with the complex network of mitochondrial membranes can be envisaged.

In support of this, LogD determination indicated values close to 4 for almost all dyes (Table S2 in supporting information) thus revealing the high hydrophobicity of the series. In comparison, the partition coefficient of PicoGreen is significantly lower (calculated LogD = 2.14) which could explain its different behaviour. To further evaluate the dyes affinity for lipidic membranes, fluorimetric titration of TP-2Bzim and TN-2Bzim was carried out with liposomes made of dioleoylphosphaditylcholine (DOPC) (Figure S9). The strong fluorescence increase observed in both cases upon addition of liposomes indicates clearly immobilization of the compounds upon binding to the membrane. Taken together our data fully support the assumption that the dyes remain sequestered in the inner membrane, which furthermore has a high content in negatively charged phospholipids that may additionally contribute to the trapping through electrostatic attraction.

Mitochondrial Membrane Potential

The mitochondrial localization of the probes is consistent with their lipophilic cationic nature which strongly suggests that their sequestration is directly dependent on m. To further examine this point, we carried out experiments with Carbonyl Cyanide m-ChloroPhenyl hydrazone (CCCP), an uncoupling reagent able to depolarize the membrane via inhibition of the oxidative phosphorylation. To this end, HeLa cells were preincubated with the dyes for 2 h and then treated with CCCP. As shown from Figure 6, TP-2Bzim and TP 1+ -2Bzim were gradually released from mitochondria and moved to the nuclear compartment that displayed a bright fluorescence signal (Figure 6 F-H, J-L). This translocation from mitochondria to nuclear compartment corroborates the strong affinity of the two dyes for duplex DNA shown in vitro and also confirms that small molecular weight compounds diffuse freely through the nuclear membrane in live cells. Interestingly, after removal of CCCP, both dyes translocated back to their initial location site i.e. mitochondria (see SI, Figure S10). This reversible shuttling from mitochondria to nuclear compartment demonstrate firstly that TP-2Bzim and TP 1+ -2Bzim are released from mitochondria when m dissipates and reversibly reintegrate their initial mitochondrial localization when m is restablished. This result reveals that the two dyes are indicators of transient variations of m that may be triggered by endogenous or exogenous factors. In particular the strong brightness of TP-2Bzim once in the nucleus could be used as efficient read-out of m perturbation (using for instance flow cytometry). This shuttling from one compartment to another without apparent disturbance of the cell function illustrates the exceptional dynamic of the cells suborganization which may accommodate probe movement. Although the trafficking of macromolecules like mRNA or proteins from the nucleus to the cytoplasm is well documented, the displacement of small molecules/drugs is less well known. Hence fluorescent "shuttling probes" may be useful tools to sense mitochondria dysfunction as well as subtle homeostasis perturbation induced by exogenous and endogenous reagents. In order to confirm the dependence of the dye response towards m variation, we preincubated HeLa cells with the probes and treated them with hydrogen peroxide that simulates oxidative stress and induces cell death by triggering the loss of the mitochondrial membrane potential. As shown from Figure S11 (SI), TP-2Bzim and TP 1+ -2Bzim relocalized to the nucleus after this treatment thereby acting as detectors of oxidative stress. In contrast, for TN-2Bzim a diffuse fluorescence was detected in the cytosol after CCCP addition similarly to what was observed in fixed cells. This indicates that the dye is also released from the mitochondria under m dissipation but does not relocalize in the nucleus in consistency with its low affinity towards DNA. Since in this case, the dye translocates from mitochondria to cytosol, the phenomenon is less easy to visualize/image. We previously reported that translocation of TP-2Bzim from mitochondria to nucleus can be triggered by visible or NIR light upon prolonged irradiation. [17][18][19] In these conditions apoptosis was induced concomitantly due to ROS production initiated by the dye excited state which further causes strong mitochondria damages and membrane permeabilization. Differently from the present study, this photoinduced translocation phenomenon is irreversible and relevant of a photosensitization effect. Nonetheless, it was of interest to control if TN-2Bzim displays the same photosensitizing activity as its TP counterpart. Hela cells were incubated with TN-2Bzim and visible light irradiation (458 nm) was carried out for 1 h. As seen from Figure 7, during the course of illumination TN-2Bzim staining shifted progressively from a typical mitochondrial pattern to a diffuse cytoplasmic labelling (Figure 7A vs 7B). Concomitantly this continuous illumination led to blebbing and cellular death through apoptosis as reported for TP-2Bzim. In this case, the diffuse cytoplasmic staining of TN-2Bzim is similar to that observed in fixed cells (Figure 3I) revealing the leakage of the dye to the cytosol upon m dissipation due to apoptosis. Figure 7C represents an enlarged field of observation after 1h of irradiation. Cells outside the irradiated area (delineated in dashed lines) display normal morphology, thus confirming that the blebbing is due to photoactivation of TN-2Bzim.

Discussion

Many dye series have been reported for labelling mitochondria but most display broad structural diversity that makes the establishment of molecular guidelines difficult. We previously developed a series of benzimidazolium vinyltriphenylamine (TPA) dyes and showed that these compounds display a pronounced mitochondria tropism in live cells and may translocate to the nuclear compartment in certain conditions. These subcellular movements can be easily followed by confocal microscopy owing to the fluorogenic property and the exquisite brightness of the TPA compounds. Using this newly synthesized series as prototype, we conducted a study based on subtle structural variations in the aim identifying the factors that influence the dye subcellular behaviour. To this end we explored the chemical space around the parent compound TP-2Bzim to generate a panel of four analogues (TP n -2Bzim, TP-1Bzim, TP 1+ -2Bzim, TN-2Bzim) differing by the number of charges, number of vinyl Bzim branches and the nature of the aromatic core. This panel enabled us to study more in depth the structure determinants involved in the mitochondriotropism, mtDNA and nuclear DNA affinity, nuclear localization and translocation phenomenon. After characterization of the dye properties in vitro (DNA affinity and fluorescence), the systematic comparison of their performances in fixed and live cells led to the following findings: -By adjusting the charge from 0 to 2, we showed that one cationic charge is enough to confer affinity for DNA in vitro, the two monocationic compounds TP-1Bzim and TP 1+ -2Bzim exhibiting similar affinities. This rule can be applied for labelling nuclear DNA in fixed cells but with a notable exception: the monobranched compound (TP-1Bzim) is not able to reach the nuclear compartment evidencing the requirement of a 2branched moiety (TP 1+ -2Bzim) for binding DNA in fixed cells. This indicates that the DNA binding affinities determined in vitro do not fully reflect the performances of dyes in fixed cells, which can be significantly modulated by specific cellular factors. This demonstrates clearly that the binding to nuclear DNA is not solely driven by electrostatics. Finally, the global charge and its distribution has strong impact on the optical properties since both absorption and quantum yield are diminished for the monocationic TP 1+ -2Bzim. In this regard the 2-branched 2+charged compound TP-2Bzim remains the best candidate for nuclear DNA staining combining exceptional brightness and binding affinity.

-The replacement of the triphenylamine (TP) core for the larger trinaphtyl (TN) core abolishes binding to DNA likely by a steric effect preventing insertion in the minor groove, which is corroborated by the incapacity of the dye to label the nuclear compartment in fixed cells. Although this modification increases remarkably the two-photon absorption, the quantum yield and the one-photon absorption are particularly low which might reflect a twisted conformation both in the fundamental and excited state resulting from steric clash between the trinapthyl core and the Bzim moiety. [16b] -In live cells, we observed that all dyes are permeant including the neutral derivative TPn-2Bzim. However, the latter displays a nonspecific cytoplasmic staining similar to that observed in fixed cells. In counterpart the three cationic dyes (TP-1Bzim, TP 1+ -2Bzim, TN-2Bzim) are all trapped in mitochondria similarly to the parent TP-2Bzim, which again indicate that 1 cationic charge is sufficient for accumulation in mitochondria. This result illustrates the power of the electrostatic gradient created through the mitochondria membrane. However, although the Nernst equation predicts that the concentration of 2+ vs 1+ compounds can be 1000 times greater in mitochondria, this is difficult to estimate from the staining intensity that might be modulated by each dye brightness as well as its intramitochondrial localization. Remarkably these results demonstrate that the DNA affinity is not playing a role in the initial trapping phenomena confirming that the mitochondria sequestration is driven essentially by the lipophilic /cationic balance as reported for most probes. [26] In this regard the determination of logD indicated that all Bzim dyes have a high liphophilicity (logD > 3), which could be determinant for efficient uptake. Taken together the data show that the ratio logD/charge is a valuable indicator whilst it is certainly modulated by other factors inherent to the dye as pointed recently. [11e, 19] -Since our dyes have high affinity for DNA, we addressed the question of internal localization of the dyes and whether they can bind mitochondrial DNA. Unexpectedly the staining is not modified in Rho (0) cells lacking mtDNA thus showing that none of the cationic dyes were able to label the mtDNA. Again, the high lipophilicity of Bzim dyes and in particular the strong hydrophobicity of the TP or TN cores can contribute to strong embodiment in lipophilic membranes which constitute barriers preventing the access to the matrix. Since even the best DNA stainers of the series (TP-2Bzim, TP 1+ -2Bzim) do not label mitochondrial DNA, this indicates that the DNA affinity is counterbalanced by the complexity of the mitochondria architecture with polarized membranes, interspace membrane and high protein/lipid anionic content of the inner membrane. In addition, although many cationic DNA binders are likely to be sequestered fully or partially by mitochondria, our observation indicate that binding to mtDNA is not systematically achieved calling for care in data interpretation. Finally other parameters can play a role both in dye trapping and intramitochondrial localization in line with our recent observation that molecular shape /volume of TPA dyes affect dramatically the passage through the plasma membrane and their subcellular localization. [19] -Finally, the relocalization of TPA-TNA dyes upon m dissipation induced by exogenous stimuli (CCCP, H2O2) indicates the strong dependence on the membrane polarization status. However, in contrast to mitochondria trapping and internal localization, the spatial redistribution is directly correlated to their cellular localization in fixed cells as translocation to nuclear compartment was observed only for the two nuclear stainers in fixed cells (TP 1+ -2Bzim, TP-2Bzim) whereas leakage to the cytosol was seen for TN-2Bzim. This reveals that a reasonable level of DNA affinity should be achieved to allow mitochondria to nuclear translocation. Interestingly, the phenomenon is reversible and the TPA dyes reintegrate their mitochondrial localization when the membrane potential is reestablished. This points out to the high dynamics of the cellular subcompartments which enable drug/small molecules movements and exchanges but has been poorly explored so far. Finally and interestingly, translocation of TPA-TNA occurs both in conditions of transient loss of m induced by physiological circumstances or of long lasting /permanent m dissipation induced by light and associated with apoptosis, which altogether make them valuable indicators of membrane potential variations.

Conclusion

The cationic character of DNA-interactive dyes confers both nuclear DNA-labelling potential and mitochondria-tropism. However, in live cells, the partitioning between the nuclear and mitochondrial compartments is governed by a number of other factors like lipophilicity and subtle differences in molecular shape and charge distribution. Hence the cellular behaviour of dyes is difficult to predict and to rationalize. For instance, Picogreen distributes between mitochondrial and nucleus whereas the Bzim dyes have exclusive subcompartment localization (nucleus vs mitochondria). In addition, the precise localization of dyes inside mitochondria is rendered difficult by the complex architecture of this subcompartment and the possible partitioning between membranes, interspace, matrix and mtDNA, which should be taken into consideration in the data interpretation. In that sense, our structure activity study focused on the Bzim dyes illustrates the challenge for finding the trade-off between the multiple requirements for a precise targeted-localization of small molecules while retaining brightness and fluorogenic properties for optimized imaging. Ultimately our study shows that targeted-fluorescent dyes represent invaluable chemical probes to study the dynamic nature of compartments, yet poorly understood, and to provide deeper understanding of intracellular trafficking of drugs and hence of their cellular activity.

Experimental Section

General experimental procedures, analysis and instrumental details are provided in the Supporting Information.

TP n -2Bzim and TP-2Bzim have been previously reported in the literature and were synthesized following the reported protocols. The spectral data were in agreement with the literature values. [16b] Synthesis of triphenylamine 1: To a solution of 4-formyltriphenylamine (100 mg, 0.37 mmol, 1eq) and and (N-methyl benzimidazole)methyltriphenylphosphonium chloride (244 mg, 0.55 mmol, 1.5 eq) in a mixture of THF/EtOH (2 mL, 1:1, v/v) was added 1,8-Diazabicyclo [5.4.0]undec-7-ene, DBU (164 µL, 1.10 mmol, 3.0 eq.). The solution was stirred overnight at room temperature in dark. Solvents were evaporated and the crude product was purified by column chromatography over silica gel (cyclohexane/ EtOAc 6:4) to afford the desired compound 1 as a yellow powder (68.

Synthesis of TP-1Bzim:

To a solution of triphenylamine 1 (13.8 mg, 0.03 mmol, 1 eq) in DMF (0.5 mL) was added MeI (0.2 mL) and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated to dryness. The crude residue was dissolved in a minimal amount of methanol and precipitated by addition of diethyl ether, affording in quantative yield the desired compound as a yellow solid (18.7 mg, quantitative yield). m.p. > 260°C.

Synthesis of aldehyde 2:

To a solution of 4,4'-bisformyltriphenylamine (50.0 mg, 165.9 µmol, 3.0 eq.) and (N-methyl benzimidazole)methyltriphenylphosphonium chloride (24.5 mg, 55.3 µmol, 1.0 eq.) in a mixture of THF/EtOH (0.82 mL, 1:1, v/v) was added 1,8-Diazabicyclo [5.4.0]undec-7-ene, DBU (16.5 µL, 111 µmol, 2.0 eq.). The solution was stirred overnight at room temperature in dark. Solvents were concentrated and the crude residue was purified by column chromatography over silica gel (cyclohexane/EtOAc 7:3) to afford the desired compound 2 as a yellow powder (20.0 mg, 84%). m.p. 115-122°C.

Synthesis of TP 1+ -2Bzim:

To a solution of aldehyde 2 (50 mg, 116.5 µmol, 1.0 eq.) and 1,2,3-trimethyl-1Hbenzo [d]imidazole-3-ium iodide (33.5 mg, 116.5 µmol, 1.0 eq.) in EtOH (4 mL) was added piperidine (23 µL, 233 µmol, 2.0 eq.). The mixture was stirred overnight at 80°C. The black solution was concentrated and the crude product was purified by column chromatography over silica gel (CH2Cl2/ EtOH 98:2) to give the desired compound (31 mg, 38%) as orange powder. m.p. > 260°C. 1 H NMR (300 MHz, DMSO) δ = 8.03 (dd, J = 6.0, 3.0 Hz, 2H, Ar-H), 7.88 -7.74 (m, 6H, Ar-H), 7.67 (dd, J = 6.0, 3.0 Hz, 2H, Ar-H), 7.63 -7.50 (m, 2H, Ar-H), 7.48 -7.34 (m, 4H, Ar-H), 7.23 -7.10 (m, 9H, Ar-H), 4.13 (s, 6H, CH3), 3.92 (s, 3H, CH3).

Synthesis of dialdehyde 3:

In dry and degassed toluene (30.0 mL) were introduced Pd(OAc)2 (30.0 mg, 125.0 µM, 5 mol%) and P(t-Bu)3 (1.2 mL, 10% in hexanes w/w, 7.0 mmol, 12 mol%). After 15 min stirring, 6-bromo-2-naphthaldehyde (2.3 g, 7.0 mmol, 2.8 eq.), 2-aminonaphthalene (500 mg, 2.5 mmol, 1.0 eq.) and Cs2CO3 (2.0 g, 6.25 mmol, 2.5 eq.). The solution was refluxed 48 h, cooled to room temperature and diluted with CH2Cl2 (100 mL). The crude mixture was filtered through a celite® pad, concentrated and purified by column chromatography over silica gel (cyclohexane/CH2Cl2 2:1 to 1:2) to afford product 3 (610.0 mg, 38%) as a yellow powder. m.p. 135-142°C. 1 H NMR (300 MHz, CDCl3) δ = 10.12 (s, 2H, CHO), 8.28 (s, 2H, Ar-H), 7.94-7.85 (m, 6H, Ar-H), 7.66 (d, J = 9.0 Hz, 2H, Ar-H), 7.54 (s, 2H, Ar-H), 7.49-7.45 (m, 4H, Ar-H)

Synthesis of TN-2Bzim:

To a solution of dialdehyde 3 (40.0 mg, 89 µmol, 1.0 eq.) and 1,2,3-trimethyl-1Hbenzo [d]imidazole-3-ium iodide (51 mg, 180 µmol, 2.0 eq.) in EtOH (4.0 mL) was added piperidine (35.0 µL, 356 µmol, 4.0 eq.). The mixture was stirred overnight at 80°C. The reaction mixture was cooled to room temperature, then the desired compound was precipitated in Et2O to afford the desired compound (27 mg; 31%) as a dark powder. m.p. > 260°C. 1 H NMR (300 MHz, DMSO) δ = 8.37 (s, 2H, Ar-H), 8.14 (d, J = 8.5 Hz, 2H, Ar-H), 8.09 -7.98 (m, 10H, Ar-H), 7.92 (d, J =9.0 Hz, 2H, Ar-H), 7.83 -7.81 (m, 1H, Ar-H), 7.71-7.62 (m, 9H, Ar-H), 7.55 -7.38 (m, 5H, Ar-H), 4.20 (s, 12H, CH3).

Figure 1 .

 1 Figure 1. Chemical structures of TP-1Bzim, TP-2Bzim, TP n -2Bzim, TP 1+ -2Bzim, TN-2Bzim.

Scheme 1 .

 1 Scheme 1. Synthesis of TP-1Bzim, TP 1+ -2Bzim and TN-2Bzim. Reagents and conditions: (a) (N-methyl benzimidazole)-methyltriphenylphosphonium chloride , DBU, THF/EtOH, r.t., 18h, 47% ; (b) MeI, DMF, 60°C, 100% ; (c) (N-methyl benzimidazole)-methyltriphenylphosphonium chloride , DBU, THF/EtOH, r.t., 18h, 84% ; (d) 1,2,3-trimethyl-1Hbenzo[d]imidazole-3-ium iodide, piperidine, EtOH, 80°C, 18h, 38% ; (e) Pd(OAc)2, P(t-Bu)3, Cs2CO3, toluene, reflux, 48h, 38%; (f) 1,2,3-trimethyl-1Hbenzo[d]imidazole-3-ium iodide, piperidine, EtOH, 80°C, 15h, 31%.

Figure 2 .

 2 Figure 2. Absorption (in blue) and fluorescence spectra (in red) of A) new compounds at 10 µM and 1 µM, respectively and B) TP-2Bzim at 10 µM and 0.25µM respectively (already described in ref 22), upon addition of DrewAT (sequence: 5'-CGCG-AAATTT-TCGCG-3') (0-20µM) in sodium cacodylate buffer 10 mM at pH 7.2, NaCl 400 mM (exc = 465 nm for TP-1Bzim, exc = 485 nm for TP n -Bzim, exc = 515 nm for TP 1+ -Bzim, exc = 525 nm for TN-2Bzim, exc = 460 nm for TP-2Bzim). C) Maximum fluorescence enhancement of the dyes in presence of drewAT in saturation conditions and affinity constants calculated from the fluorimetric titrations using nonlinear curve fitting analysis (see SI).

Figure 3 .

 3 Figure 3. Localization of dyes in fixed (A,C,E,G,I) and live (B,D,F,H,J) Hela cells, TP n -2Bzim (exc = 405 nm, em = 415-700 nm); TP 1+ -2Bzim, TP-2Bzim and TN-2Bzim (exc = 488 nm, em = 500-700 nm), fixation (3% paraformaldehyde in PBS for 15 min).Scale bar: 10 µm.

Figure 4 .

 4 Figure 4. Co-localization experiments with TP-1Bzim (A) TP 1+ -2Bzim (B) TP-2Bzim (C) and TN-2Bzim (D)exc = 488 nm, em = 500-650 nm). Hela Cells were first incubated with dyes at 2µM for 2h, then incubated with MitoTracker® Deep Red (100 nM, 45 min; exc = 633 nm, em = 650-800 nm). Scale bar: 10µM.

Figure 5 .

 5 Figure 5. Imaging of live HeLa cells with or without ddC treatment (exc = 488 nm, em = 500-800 nm). A, B, C, D) Cells were grown 4 days and stained with dyes: Picogreen® (1.5 µL/mL, 1 h), TP 1+ -2Bzim, TP-2Bzim or TN-2Bzim (2 µM, 2 h). E, F, G, H) After incubation 4 days with ddC at 20 µM, cells were stained with dyes: Picogreen® (1.5 µL/mL, 1 h), TP 1+ -2Bzim, TP-2Bzim or TN-2Bzim (2 µM, 2 h). Scale bar: 10 µm.

Figure 6 .

 6 Figure 6. Imaging of live HeLa cells under m perturbation (exc = 488 nm, em = 500-800 nm). Cells were stained with dyes at 2 µM, 2 h: CMXROS (A), TP 1+ -2Bzim (E), TP-2Bzim (I) and TN-2Bzim (M). Cells were stained with dyes at 2 µM, 2 h and incubated with CCCP at 20 µM, 20: CMXROS (B, C, D), TP 1+ -2Bzim (F, G, H), TP-2Bzim (J, K, L) and TN-2Bzim (N, O, P) recorded at 2, 10 and 20 minutes. Scale bar: 10 µm.

Figure 7 .

 7 Figure 7. Confocal fluorescence imaging of TN-2Bzim-treated living HeLa cells upon prolonged visible light irradiation (exc = 458 nm, em = 500-800 nm). Cells were pre-incubated with 2 µM TN-2Bzim for 2 h at 37°C. Imaging of TN-2Bzim A) before continuous irradiation and B) after 1 h irradiation (exc = 458 nm, irradiance 30 mW.cm -2 ). Corresponding transmission images illustrating the concomitant membrane blebbing (orange arrows) are shown below fluorescence images. Scale bar: 10µm. C) Enlarged field of observation for t= 1 h. The delineated area corresponds to the irradiated area. Scale bar: 20µm.

  1 H NMR (300 MHz, DMSO) δ 8.02 (dd, J = 6.0, 3.0 Hz, 2H, Ar-H), 7.79 (d, J = 8.5 Hz, 2H, Ar-H), 7.74 (d, J = 16.5 Hz, 1H, CH=CH), 7.67 (dd, J = 6.0, 3.0 Hz, 1H, Ar-H), 7.39 (t, J = 7.5 Hz, 4H, Ar-H), 7.33 (d, J = 16.5 Hz, 2H, CH=CH), 7.16 (m, 6H, Ar-H), 7.20 -7.12 (d, J = 8.5 Hz, 2H, Ar-H), 4.11 (s, 3H, CH3). 13 C NMR (75 MHz, DMSO) δ 150.1 (Cq), 148.7 (Cq), 146.2 (Cq), 146.2 (CH), 132.0 (Cq), 130.2 (CH), 129.9 (CH), 127.2 (Cq), 126.3 (CH), 125.4 (CH), 124.6 (CH), 120.6 (CH), 112.8 (CH), 104.7 (CH), 32.9 (CH3). HRMS (ESI+) m/z calcd for C29H26N3: 416.2138 [M-I] + , found: 416.2127.

  1 H NMR (300 MHz, CDCl3) δ = 9.84 (s, 1H, CHO), 7.94 (d, J = 16.0 Hz, 1H, CH=CH), 7.79 -7.74 (m, 1H, Ar-H), 7.72 (d, J = 8.5 Hz, 2H, Ar-H), 7.56 (d, J = 8.5 Hz, 2H; Ar-H), 7.41 -7.27 (m, 5H, Ar-H), 7.24 -7.14 (m, 5H, Ar-H), 7.11 (d, J = 8.5 Hz, 2H, Ar-H), 7.04 (d, J = 16.0 Hz, 1H, CH=CH), 3.87 (s, 3H, CH3). 13 C NMR (75 MHz, CDCl3) δ = 190.5 (CH), 152.8 (Cq), 151.1 (Cq), 146.9 (Cq), 145.9 (Cq), 143.1 (Cq), 136.2 (CH), 136.2 (Cq), 132.2 (Cq), 131.3 (CH), 129.9 (CH), 129.8 (Cq), 128.5 (CH), 126.6 (CH), 125.5 (CH), 125.4 (CH), 122.7 (CH), 122.5 (CH), 120.5 (CH), 119.2 (CH), 112.2 (CH), 109.2 (CH), 29.7 (CH3). HRMS (ESI + ): m/z calcd for C29H24N3O: 430.1920 [M+H] + , found: 430.1919.

  13 C NMR (75 MHz, DMSO) δ = 151.1 (Cq), 149.5 (Cq), 148.6 (Cq), 146.7 (Cq), 146.0 (Cq), 145.9 (CH), 142.8 (Cq), 136.0 (Cq), 135.1 (CH), 131.9 (Cq), 131.6 (Cq), 130.2 (CH), 130.0 (CH), 128.9 (CH), 128.0 (Cq), 126.3 (CH), 125.7 (CH), 125.0 (CH), 124.3 (CH), 122.0 (CH), 121.9 (CH), 121.8 (CH), 118.3 (CH), 113.3 (CH), 112.8 (CH), 110.1 (CH), 105.2 (CH), 32.9 (CH3), 29.6 (CH3). HRMS (ESI+) m/z calcd for C39H34N5: 572.2814 [M-I] + , found: 572.2815.

  , 7.38 (dd, J = 9.0, 1.5 Hz, 1H, Ar-H). 13 C NMR (75 MHz, CDCl3) δ = 192.0 (CH), 147.9 (Cq), 144.2 (Cq), 137.9 (Cq), 134.5 (Cq), 134.1 (CH), 133.3 (Cq), 131.3 (Cq), 131.0 (CH), 130.0 (CH), 129.4 (Cq), 128.1 (CH), 127.9 (CH), 127.5 (CH), 126.9 (CH), 125.9 (CH), 125.3 (CH), 125.0 (CH), 124.0 (CH), 123.5 (CH), 120.1 (CH). MS (ESI+) m/z 452.2 [M+H] + .

  , DMSO) δ = 148.3 (Cq), 146.4 (CH), 146.4 (Cq), 144.1 (Cq), 135.5 (Cq), 134.1 (Cq), 132.1 (Cq), 131.0 (Cq), 130.7 (CH), 130.7 (Cq), 130.4 (CH), 129.9 (CH), 129.5 (Cq), 127.9 (CH), 127.7 (CH), 127.3 (CH), 126.7 (CH), 126.5 (CH), 125.6 (CH), 125.1 (CH), 124.7 (CH), 124.3 (CH), 122.6 (CH), 119.7 (CH), 113.0 (CH), 107.3 (CH), 33.0 (CH3). HRMS (ESI+) m/z calcd for C52H43N5I: 864.2563: [M-I] + , found: 864.2548.

Table 1 .

 1 Linear optical properties of vinyltriarylamine derivatives in pH 7.2 cacodylate buffer with or without DrewAT and in glycerol.

	Solvent	exc
		em	[b]

Acknowledgements

We are very grateful to Dr. Marie-Noelle Soler and Laetitia Besse from the photonic microscopy platform (Institut Curie, CNRS UMS2016, INSERM U43, Université Paris-Saclay, Orsay, France) for technical assistance in the use of the Leica confocal SP5 system. We are grateful to Prof. Wiesner and Ms. Bust from Institute for Vegetative Physiology, University of Cologne, Germany and Prof. Hayashi from University of Tsukuba, Japan for providing the mitochondrial DNA-lacking HeLa rho0 cells. This work has benefited from the facilities and expertise of the Small Molecule Mass Spectrometry platform of ICSN (Centre de Recherche de Gif -www.icsn.cnrs-gif.fr) as well as the facilities of TechMedIll (UMS3286). We wish to thank Dr. Nicolas Tsapis for providing the liposomes of DOPC.

Entry for the Table of Contents

Fluorescent probes based on a triphenylamine core have been designed and synthesized as cellular imaging tools for monitoring the mitochondrial status. This study was carried out to decipher the mechanism of relocalization from mitochondria to nucleus under mitochondrial membrane depolarisation.


Dr Julien Hadoux
email: julien.hadoux@gustaveroussy.fr

Pauline Afchain

Thomas Walter

David Tougeron

Vincent Hautefeuille

Carole Monterymard

Véronique Lorgis

Frédéric Thuillier

Eric Baudin

Jean Yves Scoazec

Côme Lepage

Romain Desgrippes

FOLFIRINEC: a randomized phase II trial of mFOLFIRINOX vs platinum-etoposide for metastatic neuroendocrine carcinoma of gastroenteropancreatic or unknown origin

Keywords:

Rational and aims

Poorly differentiated neuroendocrine carcinomas (NEC) of gastroenteropancreatic (GEP) and unknown origins are rare and heterogeneous diseases. The diagnosis is often done at the metastatic stage and the prognosis is poor. The standard first-line (L1) treatment is platinum-etoposide (PE) combination chemotherapy, mostly based on retrospective studies [START_REF] Pavel | Gastroenteropancreatic neuroendocrine neoplasms: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF][START_REF] De Mestier | Digestive Neuroendocrine Neoplasms (NEN): French Intergroup clinical practice guidelines for diagnosis, treatment and follow-up (SNFGE, GTE, RENATEN, TENPATH, FFCD, GERCOR, UNICANCER, SFCD, SFED, SFRO, SFR)[END_REF]. With this regimen, RR is 40 to 70% but the median PFS is short, between 4 and 9 months [START_REF] Sorbye | Predictive and prognostic factors for treatment and survival in 305 patients with advanced gastrointestinal neuroendocrine carcinoma (WHO G3): The NORDIC NEC study[END_REF][START_REF] Walter | Poorly differentiated gastro-entero-pancreatic neuroendocrine carcinomas: Are they really heterogeneous? Insights from the FFCD-GTE national cohort[END_REF]. Disease progression almost always occurs during or just after treatment and median overall survival (OS) is only about 12-15 months for GEP NEC with similar efficacy of either cisplatin or carboplatin [START_REF] Sorbye | Predictive and prognostic factors for treatment and survival in 305 patients with advanced gastrointestinal neuroendocrine carcinoma (WHO G3): The NORDIC NEC study[END_REF][START_REF] Walter | Poorly differentiated gastro-entero-pancreatic neuroendocrine carcinomas: Are they really heterogeneous? Insights from the FFCD-GTE national cohort[END_REF] and of either oral or intravenous etoposide [START_REF] Frizziero | Carboplatin in Combination with Oral or Intravenous Etoposide for Extra-Pulmonary, Poorly-Differentiated Neuroendocrine Carcinomas[END_REF][START_REF] Ali | Intravenous versus oral etoposide: efficacy and correlation to clinical outcome in patients with high-grade metastatic gastroenteropancreatic neuroendocrine neoplasms (WHO G3)[END_REF]. After progression, only 40 to 45% of patients will receive a second-line (L2) chemotherapy which will include 5fluoro-uracil (5FU) or capecitabine and irinotecan (FOLFIRI [START_REF] Hentic | FOLFIRI regimen: an effective second-line chemotherapy after failure of etoposide-platinum combination in patients with neuroendocrine carcinomas grade 3[END_REF]) or oxaliplatin (XELOX, FOLFOX [START_REF] Hadoux | Post-firstline FOLFOX chemotherapy for grade 3 neuroendocrine carcinoma[END_REF]) or dacarbazine/temozolomide [START_REF] Couronne | Post first-line dacarbazine or temozolomide in neuroendocrine carcinoma[END_REF]. This second-line treatment can provide about 30% of RR and a median PFS of 4 months [START_REF] Walter | Poorly differentiated gastro-entero-pancreatic neuroendocrine carcinomas: Are they really heterogeneous? Insights from the FFCD-GTE national cohort[END_REF][START_REF] Hentic | FOLFIRI regimen: an effective second-line chemotherapy after failure of etoposide-platinum combination in patients with neuroendocrine carcinomas grade 3[END_REF][START_REF] Hadoux | Post-firstline FOLFOX chemotherapy for grade 3 neuroendocrine carcinoma[END_REF]. Taken together, these data indicate a major medical need for improving NEC treatments.

Since the first description, thirty years ago, of the PE combination efficacy for what was called "anaplastic neuroendocrine carcinomas" and referred nowadays to NEC by Moertel and collaborators, no change has been made to this standard of care [START_REF] Moertel | Treatment of neuroendocrine carcinomas with combined etoposide and cisplatin. Evidence of major therapeutic activity in the anaplastic variants of these neoplasms[END_REF]. Only one randomized phase II trial have compared the efficacy of the cisplatin -irinotecan (PI) combination to the standard PE regimen. This phase II enrolled 66 patients and was terminated prematurely following interim analysis showing equivalent efficacy. Indeed, the objective RR were similar in both arm (42.4%), the median PFS was 6.4 months in the PE arm and 5.8 months in the PI arm, respectively (p=0.81), and the median OS was 11.3 months and 10.2 months, respectively, (p=0.37) [START_REF] Zhang | Etoposide and cisplatin versus irinotecan and cisplatin as the first-line therapy for patients with advanced, poorly differentiated gastroenteropancreatic neuroendocrine carcinoma: A randomized phase 2 study[END_REF]. A single arm phase II study evaluated PE intensification with the addition of paclitaxel and found an objective RR of 53%, a median PFS of 7.5 months and a median OS of 14.5 months which led the authors to conclude to the absence of higher efficacy as compared to the standard PE regimen [START_REF] Hainsworth | Phase II trial of paclitaxel, carboplatin, and etoposide in advanced poorly differentiated neuroendocrine carcinoma: a Minnie Pearl Cancer Research Network Study[END_REF]. Other studies are almost all retrospective and have reported equivalent efficacies of either PE or PI, except the Yamaguchi et al. study suggesting higher efficacy of PI regimen but this study was not randomized, the studies of firstline chemotherapy in NEC are summarized in Table 1.

Although all studies on second-line treatment of metastatic NEC of GEP or unknown origin were retrospective, they have suggested that both irinotecan and oxaliplatin, in combination with 5FU can have anti-tumor effect in NEC [START_REF] Hentic | FOLFIRI regimen: an effective second-line chemotherapy after failure of etoposide-platinum combination in patients with neuroendocrine carcinomas grade 3[END_REF][START_REF] Hadoux | Post-firstline FOLFOX chemotherapy for grade 3 neuroendocrine carcinoma[END_REF]. In the last decade, the FOLFIRINOX triplet chemotherapy regimen, combining 5FU, oxaliplatin and irinotecan, has shown significant efficacy in several digestive cancers such as pancreas [START_REF] Conroy | FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer[END_REF] or colorectal adenocarcinoma [START_REF] Ychou | A randomized phase II trial of three intensified chemotherapy regimens in first-line treatment of colorectal cancer patients with initially unresectable or not optimally resectable liver metastases. The METHEP trial[END_REF]. Tolerance of this regimen has improved over the years with better tolerability that has led to the development of the mFOLFIRINOX regimen [START_REF] Lambert | Current status on the place of FOLFIRINOX in metastatic pancreatic cancer and future directions[END_REF]. mFOLFIRINOX could be a good L1 treatment in metastatic GEP NEC because: (i) Oxaliplatin, irinotecan and 5FU have anti-tumor effect in metastatic GEP NEC [START_REF] Walter | Poorly differentiated gastro-entero-pancreatic neuroendocrine carcinomas: Are they really heterogeneous? Insights from the FFCD-GTE national cohort[END_REF][START_REF] Hentic | FOLFIRI regimen: an effective second-line chemotherapy after failure of etoposide-platinum combination in patients with neuroendocrine carcinomas grade 3[END_REF][START_REF] Hadoux | Post-firstline FOLFOX chemotherapy for grade 3 neuroendocrine carcinoma[END_REF]; (ii) triplet with a potential high RR could be efficient in these chemosensitive cancers; (iii) the degradation of PS following tumor progression during/after L1 treatment makes access to a second-line uncertain which argue for the use of an aggressive L1 treatment; (iiii) administration on a one-day outpatient basis (day hospital), as well as acceptable adverse events, could have an impact on quality of life in these patients with a poor prognosis. With the PRODIGE 69-FOLFIRINEC trial hypothesize that the mFOLFIRINOX triplet may improve the prognosis of patients with metastatic NEC from GEP or unknown primary.

Few data are available on predictive factors of L1 chemotherapy efficacy in metastatic NEC which are subdivided in 2 main pathological subtypes; eg. small cell NEC (SCNEC) and large cell NEC (LCNEC). These two subtypes are treated with the same PE regimen, whatever the primary tumour site, although overall response rate (ORR) seem to differ between SCNEC (about 50 to 70%) and LCNEC (about 30-50%) in lung and pancreas NEC retrospective series [START_REF] Zhuo | The Prognostic and Therapeutic Role of Genomic Subtyping by Sequencing Tumor or Cell-Free DNA in Pulmonary Large-Cell Neuroendocrine Carcinoma[END_REF][START_REF] Derks | Genetic subtypes of large cell neuroendocrine carcinoma (LCNEC) to predict response to chemotherapy[END_REF][START_REF] Tanaka | Pancreatic neuroendocrine carcinoma G3 may be heterogeneous and could be classified into two distinct groups[END_REF][START_REF] Treut | Multicentre phase II study of cisplatin-etoposide chemotherapy for advanced large-cell neuroendocrine lung carcinoma: the GFPC 0302 study[END_REF]. These data have led to the choice of stratifying the FOLFIRNIEC trial according to pathological subtypes. Few studies have reported molecular profiles of NEC with a perspective of personalized treatment. Previous studies have mainly focused on BRAF mutation in colon NEC and the efficacy of the dabrafenib-trametinib combination [START_REF] Dizdar | BRAFV600E mutation: A promising target in colorectal neuroendocrine carcinoma[END_REF][START_REF] Capdevila | Epigenetic EGFR Gene Repression Confers Sensitivity to Therapeutic BRAFV600E Blockade in Colon Neuroendocrine Carcinomas[END_REF][START_REF] Klempner | BRAFV600E Mutations in High-Grade Colorectal Neuroendocrine Tumors May Predict Responsiveness to BRAF-MEK Combination Therapy[END_REF], contrary to what is observed in colon adenocarcinoma, which has been suggested to be related to an epigenetic silencing of the epidermal growth factor receptor in colon NEC [START_REF] Capdevila | Epigenetic EGFR Gene Repression Confers Sensitivity to Therapeutic BRAFV600E Blockade in Colon Neuroendocrine Carcinomas[END_REF]. Moreover, little is known on the putative predictive biomarkers to immunotherapy efficacy in NEC of GEP and unknown origin. Tumour mutational burden (TMB) is between 8.6 and 10.5 mutations/megabase in NEC of the lung [START_REF] George | Comprehensive genomic profiles of small cell lung cancer[END_REF][START_REF] Rekhtman | Next-Generation Sequencing of Pulmonary Large Cell Neuroendocrine Carcinoma Reveals Small Cell Carcinoma-like and Non-Small Cell Carcinoma-like Subsets[END_REF] but no data exist for GEP and unknown origin NEC. In addition MLH1 and PMS2 loss of expression by immunohistochemistry mostly due to MLH1 promoter methylation (dMMR phenotype) have been reported in 12.4% tumoral samples of a series of 89 GEP NEC and mixed neuroendocrine neoplasms [START_REF] Sahnane | Microsatellite unstable gastrointestinal neuroendocrine carcinomas: a new clinicopathologic entity[END_REF]. In an attempt to expand knowledge on molecular alterations in NEC of GEP and unknown origin, the FOLFIRINEC-PRODIGE 69 phase II trial is associated with a "real time" translational study which will establish the tumor molecular profile of each participating patients for whom tumoral sample is available.

Study design

The PRODIGE 69-FOLFIRINEC study is a national, multicentre, prospective, openlabel, randomized and trial comparing the efficacy of mFOLFIRINOX versus PE regimen for the treatment of patients with metastatic NEC of GEP and unknown origin associated with a molecular profiling for therapeutic targets and biomarkers identification (Figure 1). Eligible patients will be stratified according to ECOG PS (0 vs 1), Ki67 (<55% vs ≥55%) and pathological subtype (small cell vs large cell or unknown) and then randomly assigned (1:1) to either standard regimen arm with platinum (cisplatin 100 mg/m² day 1 or carboplatin AUC 5 day 1, according to physician's choice) and etoposide (100 mg/m² intravenous (IV), day 1, 2 and 3) administered every 21 days for 6 to 8 cycles (24 weeks maximum) or experimental treatment arm with mFOLFIRINOX (oxaliplatin 85 mg/m² IV + irinotecan 180 mg/m² IV + LV5FU2 2400 mg/m² without 5 FU bolus) administered every 14 days for 12 cycles (24 weeks maximum).

Main inclusion criteria in the PRODIGE 69-FOLFIRINEC trial are patients over 18 years, ECOG PS 0 or 1, with a metastatic NEC or high grade MiNEN with a NEC component ≥ 30%, of GEP or unknown origin, whatever the pathological subtype (small cell or large cell or non-small cell or unknown/indetermined) (Table 2). Grade 3 well-differentiated neuroendocrine tumors according to WHO 2017 classification are not eligible. Patient must not have received any prior therapy for metastatic disease. Patient with asymptomatic brain metastases or under stable corticosteroid doses for at least 2 weeks before randomization can be included; otherwise, radiation therapy prior to inclusion is required in case of symptomatic brain metastases. Patient must have adequate haematology parameters (neutrophil count ≥ 1.5x10⁹/L, platelet ≥ 100x10⁹/L and hemoglobin > 8 g/dL), a creatinine clearance above 30 ml/min (Cockroft & Gault formula) and adequate liver function (total bilirubin ≤ 1.5N, AST/ALT ≤ 2.5N, or AST/ALT ≤ 5N in case of liver metastases). All patients must undergo dihydropyrimidine dehydrogenase (DPD) deficiency screening and cannot be included in case of uracilemia ≥ 16 ng/mL. Other standard exclusion criteria are applied: pregnancy, history of recent malignancy, active HIV or viral hepatitis and any active or suspected acute or chronic uncontrolled disease that would impair study participation.

The radiological assessment will be performed at baseline (within a maximum of 3 weeks before inclusion) using a TAP computed tomography (CT) scan (or magnetic resonance imaging (MRI) of the abdomen plus chest CT-scan in case of contrast medium allergy), and the same procedure (CT and/or MRI) will be repeated every 8 weeks until tumor progression or death. Radiological tumor assessment will be performed according to the RECIST v1.1 criteria. Brain imaging is required at baseline either by CT-scan and/or MRI. Imaging of brain lesions by CT-scan and/or MRI is required every 8 weeks (+/-1 week) during treatment and follow-up if present at baseline.

Study endpoints

The primary endpoint is the median PFS. PFS is defined as the time interval between date of randomization and date of the first radiological progression (according to RECIST 1.1) or death due to any cause, whichever occurs first, according to the investigator. Patient alive without progression will be censored at date of last follow-up visit.

The secondary endpoints are:

-centralized PFS by independent reviewed -OS which is defined as the time between date of randomization and date of death (whatever the cause). Patients alive will be censored at date of last news.

-Best objective RR which is defined as the proportion of patients with an objective response (complete response (CR) + partial response (PR)) at any evaluation during the treatment, according to RECIST 1.1 and centralized review.

-Safety which is defined as the percentage of patients who experienced toxicities and grading of these toxicities according to NCI-CTC V4. Toxicities will be presented as the number of patients presenting at least one toxicity by maximum grade.

-Dose reductions and dose intensity which are defined as the number of treatment cycles, the dose received and the percentages of actual dose received as compared to the theoretical dose will be described, as well as the percentage of patients with at least one dose modification/reduction or at least one postponement of chemotherapy.

-Quality of life assessed by the EORTC QLQ-C30 and EQ-5D-5L questionnaires.

-Exploratory analyses (see "biomarkers analyses"). The predictive value of each molecular alterations will be evaluated using correlation with objective RR, PFS and OS, in both arms.

Ethical considerations

This study is sponsored by the Fédération Francophone de Cancérologie Digestive

Statistical methods

For the primary endpoint, the median PFS will be calculated among patients who have received at least one dose of chemotherapy, whatever the dose and the treatment (modified intent-to-treat). Median PFS will be given for each treatment arm with their two-sided 95% confidence interval. The Kaplan-Meier method (Kaplan and Meier, 1958) will be used to estimate median and curves will be plotted. Log-rank test will be used to compare the 2 treatment arms. Hazard ratios will be calculated using Cox proportional model (Cox, 1984). Proportionality (Schoenfeld residual methods) and linearity (Martingale residuals) hypotheses will be checked.

The hypothesis of the PRODIGE 69-FOLFIRINEC trial is that mFOLFIRINOX could increase median PFS from 5 months in the control arm (PE) to 7.5 months in the experimental arm (H1).

With a two-sided risk alpha of 5% and a power of 80%, 203 events (radiographic progression or death) are required to demonstrate a median PFS difference of 2.5 months (HR=0.67). With 24 months of follow-up, an inclusion rate of 5 patients/month and a lost-tofollow-up rate of 5%, 218 patients will be randomized.

An intermediate analysis is planned at 50% of events (102 radiographic progression or death). The intermediate analysis is planned in order to show efficacy at an early stage (rejection of H0) or futility (accept H0). The p-values will be calculated using the O'Brien-Fleming function based on the real number of events.

As G3 NEC is a rare disease, 48 centers will be open to recruitment throughout France.

Biomarker analysis

This study includes a real-time, centralized molecular profiling of the tumor consisting of immunohistochemistry (IHC) markers (PD-L1 (Programmed death-ligand 1), Rb (retinoblastoma protein), TP53, MLH1 (MutL Homolog 1)) and a targeted next generation sequencing (NGS) panel of 161 genes (Oncomine Comprehensive assay V3, ThermoFisher © , Waltham, Massachusetts, US) associated with the determination of mutational tumor burden (TMB) and microsatellite instability status (MSI). This molecular profile will be reviewed by a molecular tumor board and the report will be sent to the investigator, together with the molecular profile results, within 2 months of tumor sample submission for informing further line treatment-decision making in the case of targetable alterations.

Conclusion

PRODIGE 69 -FOLFIRINEC is designed to challenge the standard platinum-etoposide combination chemotherapy with mFOLFIRINOX for the treatment of patients with NEC of GEP and unknown origin. The associated translational study aims at identifying biomarkers of responses in these patients and to draw the mutational landscape of these tumors with the goal to find targets for personalized medicine. The first patient was included on October 20, 2020, the end of inclusion is scheduled for the end of 2024. Legend: GEP = gastroenteropancreatic, UK = unknown primary, HN = Head and neck primary, GU = Genitourinary primary, PFS = progressionfree survival, OS = overall survival, NR = Not reported  QTc interval > 450 msec for male and > 470 msec for female at EKC.

 K+ <lower limit of normal (LLN), Mg²+ < LLN, Ca²+ < LLN

(

  FFCD) and Dijon University hospital. PRODIGE 69-FOLFIRINEC has been authorized by the French medicines agency (Agence Nationale de Sécurité du Médicament et des produits de santé, ANSM) on March 23, 2020. It was submitted and approved (July 20, 2020) by the ethics committee (Comité de protection des personnes, CPP). This trial is registered on the European Union Clinical Trials Register (EudraCT no. 2019-001013-16) and on the clinicaltrials.gov website (NCT04325425). The study complies with the Declaration of Helsinki and the principles of Good Clinical Practice guidelines.



  Grade 3 well differentiated neuroendocrine tumor according to WHO 2017 classification  Symptomatic brain metastases*. Previously treated by chemotherapy or targeted therapy  History or know hypersensitivity to any of the study chemotherapy agents, or their excipients.  Known or historical active infection with HIV, or known active viral hepatitis  Pre-existing permanent neuropathy (NCI CTC V4.0 grade ≥2)  Known Gilbert's syndrome  Pregnant women or breastfeeding mother  History of prior malignancy, in the three years before randomization except for cured non-melanoma skin cancer and cured in situ cervical carcinoma  Active or suspected acute or chronic uncontrolled disease that would induce excess risk associated with study participation. Patient under guardianship and/or deprived of his/her freedom  Partial or complete Dihydropyrimidine Dehydrogenase deficiency (uracilemia ≥ 16 ng/mL)  Severe renal impairment (creatinine clearance less than 30 mL/min,according to Cockroft and Gault Formula)

Table 1 : First-line chemotherapy results in gastroenteropancreatic poorly differentiated neuroendocrine carcinoma

 1

	Authors	n patie nts	Study design	Primary		Chemotherapy regimen	response rate (%)	median PFS (months)	median OS (months)
	Zhang et al [11] 66 33 33	Monocentric randomized phase II	GEP(88%), UK(12%)	Etoposide/cisplati n Irinotecan/cisplatin 44 44	6.4 5.8	11.3 10.2
	Hainsworth et al [12]	78	Multicentric monoarm Phase II	UK(62%), lung(9%)	GEP(19%),	Etoposide/carbopl atin/paclitaxel	53	7.5	14.5
	Kulke et al [26] 4	Monocentric phase II	GEP (84%), UK (11%), lung (5%)	Irinotecan/cisplatin 25	4.5	11.4
	Sørbye et al & ali et al [3,6]	252
		129	Multicentric retrospective	GEP (69%), UK (31%)	Etoposide/cisplati n	31	4	12
		67				Etoposide/carbopl atin	30	4	11
	Yamaguchi et al [27]	258 160 46	Multicentric retrospective	GEP (100%)		Irinotecan/cisplatin 50 Etoposide/cisplati n 28	5.2 4	13 7
	Walter et al [4] 253	Multicentric retrospective	GEP (80%) UK (20%)	Etoposide/platinu m	50	6.2	11.6
	Frizziero et al [5]	98	Bicentric retrospective	GEP (54%), UK (23%), GU (21%)	Etoposide/carbopl atin	48	5.8	11.6
	Ramella et al [28]	27	Bicentric retrospective	GEP(64%), UK(21%)	Irinotecan/cisplatin 46.4	3.7	11.7
	Lokesh et al [29]	114	Monocentric retrospective	GEP (33%), lung(26%), GU (15%), HN (14%), UK(9%)	Etoposide/platinu m	24	NR	11
	Yoon et al [30] 64	Monocentric retrospective	GEP (87,5%), UK(12,5%)	Etoposide/cisplati n	28	3.5	NR

Table 2 : Main inclusion and exclusion criteria Inclusion criteria

 2 Absolute neutrophil count ≥ 1.5x10 9 /l, platelet ≥ 100x10 9 /l and hemoglobin > 8 g/dl  Total bilirubin ≤ 1.5N, AST ≤ 2.5N, ALT≤ 2.5N or AST and ALT ≤ 5N in case of liver metastases.

	 Poorly differentiated neuroendocrine carcinoma or high grade MiNEN with a NEC
	component ≥30%
	 Small cell or large cell or non-small cell or unknown/undetermined subtype
	 gastro-entero-pancreatic or unknown origin
	 Metastatic disease
	 First-line treatment
	 At least one measurable lesion according to RECIST 1.1 guidelines (CT-scan)
	 Age ≥ 18 years
	 ECOG Performance Status ≤ 1 (Appendix 4)
	 Available tumor block
	

We thank all the research and clinical networks involved: GERCOR, UNICANCER, FFCD, ENDOCAN-RENATEN.

We thank all the participating centers research and clinical teams and the patients.

Conflict of interests:

None

Funding

The study is funded by the French National Cancer Institute (PHRC-K18 139).

Source of support

Supported by Clinical Research Hospital Program grants (PHRC-K 2018) from the French ministry of health (PHRC-K18 139).

Aknowledgment

Lila Gaba, Marie Moreau, Sascha Moccozet, Laethicia Ndong, Caroline Choine, Jaïque Cario, Charlène Barreaux, and all the clinical research associate and staff from CHU de Dijon & Fédération Francophone de Cancérologie Digestive (FFCD) Ludovic Lacroix, Corinne Brandy, Leslie Girard, Zsofia Balogh, Malika Abdi and all the Gustave Roussy personalized medicine and biopathology staff for the translational study: *Patient with asymptomatic brain metastases or under stable corticosteroid doses for at least 2 weeks before randomization can be included.