extfieldsofstudy: Chemistry Medicine

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Second messenger molecules have a limited spread in olfactory cilia

Olfactory responses in the cilia of olfactory receptor cells last for longer than 10 s, which cannot be explained by free diffusion of second messengers. Takeuchi and Kurahashi show that these signaling molecules have a limited spread and remain at the site of generation for a long time.

Introduction
Olfaction starts at the sensory cilia of olfactory receptor cells (ORCs). This site mediating the sensory transduction exhibits a fine cylindrical structure with a diameter of 100-200 nm and a length of several 10s of micrometers (Usukura and Yamada, 1978;Lidow and Menco, 1984). All elements needed for signal conversion are found here: olfactory receptor protein, G protein, adenylyl cyclase, and cyclic nucleotide-gated (CNG) and Ca 2+ -activated Cl (Cl (Ca) ) channels. Through signal transduction, cAMP and Ca 2+ play central roles as second (and third) messengers within a narrow space (Schild and Restrepo, 1998;Kaupp and Seifert, 2002;Kleene, 2008;Frings, 2009). Therefore, the kinetics of the odorant response is determined by the lifetimes of active forms of proteins and the production, diffusion, and extrusion/degradation of second messenger molecules.
So far, however, there is limited information regarding the time course of active second messengers, mostly because of experimental difficulties involved in studying such narrow spaces and fine structures. Moreover, several features of olfac-Electrophysiology Ciliary currents were monitored with the whole-cell (WC) recording configuration (Hamill et al., 1981;Takeuchi and Kurahashi, 2008). The membrane potentials were corrected for by the liquid junction potential at the pipette tip (4 mV; Kurahashi, 1989). The culture dish was mounted on a laser scanning microscope (LSM 510,Axiovert 200M;Zeiss) for local photolysis or a Nikon TMD for diffuse UV photolysis. Patch pipettes (resistance 10-15 MΩ) were made of borosilicate tubing Kurahashi, 2002, 2003;Takeuchi et al., 2009Takeuchi et al., , 2013. The recording pipette was, unless otherwise indicated, filled with a Cs solution (in mM): 119 CsCl, 1 CaCl 2 , 5 EGTA, 10 Hepes, 0.0005% phenol red, and 1 caged cAMP (EMD Millipore). For caged Ca experiments, 1 mM CaCl 2 and 3 mM DM-nitrophen (EMD Millipore) were added to a pipette solution containing (in mM) 119 CsCl, 10 Hepes, and 0.0005% phenol red without adding EGTA. Data were sampled with pClamp v.10 (Molecular Devices) at 10 kHz after low-pass filtering at 2 kHz. For curve drawings, data were low-pass fast Fourier transformation-filtered at 0.02 kHz. Throughout the cell experiments, the holding voltage was −54 mV with correction for junction potentials, and care was taken to not cause saturation of response, especially when we looked at the amplification and/or adaptation. The results were analyzed by an offline computer and plotted using Microcal Origin 8.6 software (OriginLab). For curve drawings, data sampled at 1/16 kHz were used in whole cilia experiments. Data are presented as mean ± SD for the number of experiments indicated. The unpaired t test in Origin software was used for Fig. 2 D (normal and 0 Ca 2+ ) as well as Fig. 2

(E and F).
Photolysis of caged compound in cilia Caged cAMP was dissolved in DMSO (Takeuchi and Kurahashi, 2002, 2008, and caged Ca was dissolved in 10 mM CsOH (Takeuchi et al., 2009(Takeuchi et al., , 2013. Both stock solutions were then stored at −20°C in complete darkness for up to 180 d. Before each experiment, the stock was diluted to a Cs solution as described above. After the establishment of the WC recording configuration, caged compounds were introduced to the cell interior by free diffusion. The whole cilia of an ORC were stimulated with an epifluorescent UV system as previously reported (Takeuchi and Kurahashi, 2002). The UV component from a 100-W xenon lamp was used as the UV source. A computer-regulated magnetic shutter controlled the timing and duration of the UV light. The light intensity was set by a neutral-density wedge filter that was controlled by a pulse motor.
For local uncaging on a single cilium, we used the LSM system as previously reported (Takeuchi and Kurahashi, 2008). An 80-mW UV laser beam (argon laser, λ = 351, 364 nm; Coherent) was used for the photolysis. Throughout the experiments, the UV intensity was regulated by the transmission parameter of an acousto-optic tunable filter device. The region of interest (ROI) function of the LSM system was used for the spatially restricted stimulation as mentioned previously (Takeuchi and Kurahashi, 2008). Colored indicators in figures show the 2D image of light strength constructed by the Gaussian laser beam. The ROI-selected edge corresponds with 50% intensity. Red in the indicator represents 99-100%; orange, 75-99%; yellow, 50-75%; green, 25-50%; and blue, 0.02-25% (Takeuchi and Kurahashi, 2008). The laser beam moves on the x and y axes with the raster scan. During the current recording, the timing and duration of the actual laser irradiation were detected using a UV-photodiode (G6262; Hamamatsu Photonics). Because the sensitivity of the G6262 photodiode to the UV component transmitted via the optical fiber for the UV region was still low, a 488-nm laser was added for detection. This 488-nm laser beam alone did not cause photolysis when examined with cells. For double-pulse stimulation, we continuously used the scan mode and bleach mode.
Local double-pulse protocol Movements of cAMP and Ca 2+ along the ciliary cytoplasm were monitored based on the current amplification and adaptation, respectively ( Fig. 1, A and B). The nonlinear summation caused by cytoplasmic cAMP has quantitatively been well described (Lowe and Gold, 1993;Takeuchi and Kurahashi, 2003). The ciliary adaptation is completely abolished when external Ca 2+ is omitted (Kurahashi, 1990) or when a strong Ca 2+ chelator is introduced into the cytoplasm (Kurahashi and Shibuya, 1990). Furthermore, it has been broadly known that Ca 2+ entry through the CNG channel regulates a feedback modulation of CNG channel (Kurahashi, 1990;Kurahashi and Shibuya, 1990;Chen and Yau, 1994;Balasubramanian et al., 1996;Kurahashi and Menini, 1997;Leinders-Zufall et al., 1997;Kleene, 1999;Bradley et al., 2001Bradley et al., , 2004Munger et al., 2001;Boccaccio et al., 2006;Stephan et al., 2011;De Palo et al., 2012;Li et al., 2016). To obtain spatial information, we used sequential double short-pulse UV stimuli at two distinct points along a single cilium (Fig. 1, A and B). Short-pulse (<1-s) stimulations were used because in such conditions, adaptations are mainly controlled by a Ca 2+ feedback to the CNG channel (Kurahashi and Menini, 1997). With long and step stimulations, other mechanisms may be added to the adaptive phenomenon (e.g., Leinders-Zufall et al., 1999).
When a conventional raster scan in the LSM system was used, the interstimulus interval varied depending on the distance between the two stimulus points (Fig. 1 C). To fix the parameters, in this study, we set five ROIs in the scan image (ROI1-5; Fig. 1, C, E, F, H, and I). The y axis of ROI1 was designated as the upper limit (•, x0, y ROI1 ), and that of ROI5 as the lower limit (■, x ROI5 , y ROI5 ) of the scan, both of which were set outside of the cilium. The first UV stimulation was applied at ROI2 with a time series (scan) mode in LSM510. ROI3 was the second stimulus point with a short distance and ROI4 with a longer distance.
During the raster scan, the position of the laser beam moved unidirectionally from the left to the right ( Fig. 1 C, right purple arrows) and from the top to the bottom in the scan area ( Fig. 1, C, E, F, H, and I, black dotted area). The scan time was determined by the scan speed (pixel dwell) and the position of the ROIs (co-ordinates x and y; Fig. 1, C, E, F, H, and I, blue dotted area). The laser was switched on only when it positioned itself at the ROI. Fig. 1 (D-F) shows a double-pulse protocol with ROI2 and ROI3. During the first scan, ROI1, ROI2, and ROI5 were illuminated by the UV laser beam, but among them, ROI1 and ROI5 were unrelated to the stimulus to the cilium. The dark yellow arrow indicates the delay from the start of scan ( Fig. 1, C, D, and G). After ROI2 was illuminated, the position of the laser system moved to the end of scan area with the raster scan ( Fig. 1, C and E, ■). Immediately, the laser position moved to the y axis of ROI3 (purple arrow in Fig. 1 E). After the scan started from • (x0, y ROI3 ), ROI3 was illuminated by UV ( Fig. 1 F, inside of purple full circle). The sum of time from ROI2 to the terminal point and the scan time from • (x0, y ROI3 ) to ROI3 equaled the interstimulus interval of the double-pulse protocols in Fig. 1 D (gray arrow).
To extend the distance between two ROIs, we set ROI4 instead of ROI3 as the second stimulus position (Fig. 1 G). After ROI2 was illuminated and the laser beam reached the terminal, the laser position returned to the y axis of ROI4 ( Fig. 1 H, purple arrow), and then ROI4 was illuminated ( Fig. 1 I, inside of purple full circle). With these protocols, the interstimulus intervals between double pulses (ROI2-ROI3 and ROI2-ROI4) were kept constant, although the distances of ROIs were different. A slight time difference between the processes (ROI5 to the position of ROI3 and ROI5 to the position of ROI4) could be ignored in the experimental time resolution.

Ca imaging in a capillary
To visualize the change in [Ca 2+ ] i in the glass capillary, Fluo-4 was used as a Ca 2+ indicator. Fine glass capillaries were made of borosilicate tubing with a filament (outer diameter, 1.5 mm; Hilgenberg GmbH) by using an electrode puller designed for making the fine pipette to be used for intracellular recording (P2; Narishige Scientific Instruments). The LSM system equipped with a Fluor (differential interference contrast) 100×/1.3-NA (oil immersion) objective lens and an argon laser beam (λ = 458 nm; Coherent) was used for visualization of [Ca 2+ ] i by Fluo-4. Fluo-4 was dissolved in DMSO and stored at −20°C under complete darkness. The stock was diluted by a Cs pipette solution including 3 mM DM-nitrophen, 1 mM CaCl 2 , and 0 EGTA before each experiment, and the solution was put into the fine pipette. The pipette was then set on the holder of a micromanipulator, and the pipette tip was slowly approached onto the glass coverslip that formed the bottom of a Petri dish filled with normal Ringer's solution. The tip region of the pipette was bent along the surface of the coverslip such that the tip-to-taper region was situated uniformly on the focal plane. Argon lasers (λ = 351, 364 nm; Coherent) in LSM were used for photolysis.
Digital random walk with membrane-binding site Molecular diffusions in the absence and presence of binding sites on the cytoplasmic site of the plasma membrane were simulated using a digital model coded in Microsoft Visual Basic.NET 2008. A pixel imitating a single molecule situated in 3D space randomly moved (Random function in VB.net) within a long pipe that imitated a cilium or neuronal process; the diameter of the pipe was variable. In one time step, the pixel could randomly choose 1 of 27 pixels (3D) present at and surrounding the current position. A single step was defined as 10 nm. Therefore, the spatial resolution with this model was 20-nm cubes. On the cytoplasmic surface of the plasma membrane, the pixel could proceed only to the free space without penetrating the membrane. In the model, the pipe actually had a square shape in the cross section, but the walking distance from the center to the edge was the same for any angle. Therefore, the shape of the pipe was equivalent to a circular cylinder. The time resolution of the step movement was determined by the computer clock, but the theoretical time was inversely and statistically obtained from the relation between the time and variance (σ 2 ) of the moving distance. Initially, the time was defined such that the global molecular movements become ∼250 µm 2 /s (σ 2 /s). Data for D were precisely obtained after execution of tasks. When the membrane had binding sites, the pixel stayed on the cytoplasmic surface of the plasma membrane for a preconditioned period, which was equivalent to the bound time (stickiness).

Response time course and recovery from adaptation
We roughly estimated spatiotemporal dynamics of second messengers in cilia from the odorant-and cAMP-induced responses in the current response obtained with the WC recording configuration ( Fig. 2 A). Mainly, cAMP reduction was estimated from the off-kinetics of the odorant-and cAMP-induced responses, especially when Ca 2+ -dependent adaptation and the Cl − component were omitted in 0-added Ca 2+ conditions (Kurahashi, 1989). Ca 2+ dynamics was estimated from the time course of the Ca 2+ -induced current and recovery from the adaptation.
Even with a brief pulse (e.g., 100 ms) of odorant or cAMP, we found that the response lasted for several seconds (see also Takeuchi and Kurahashi, 2002). In extreme cases, the lifetime was >10 s (Fig. 2 B; see also Kurahashi, 1989;Takeuchi and Kurahashi, 2002). If cAMP diffused freely, the cytoplasmic cAMP concentration in the cilia would drop immediately, owing to molecular diffusion from the proximal part of cilia to the dendrosomatic area. It has been known that one-dimensional cAMP diffusion is considerably rapid. The SD calculated from its diffusion coefficient (D cAMP ) is 19 µm/s (D cAMP = 270-370 µm 2 /s; Chen et al., 1999); the value is already longer than the total length of newt olfactory cilia (∼10 µm). D Ca is very similar to D cAMP as described earlier.
Furthermore, we found that the lifetimes of cAMP-and Ca 2+ -induced responses were gradually prolonged during recordings after the establishment of the WC recording configuration ( Fig. 2, B and C). The half decay time of the cAMP-induced response was 0.18 ± 0.12 s (n = 3) 3 min after the establishment of the WC recording configuration ( We do not know the precise mechanism of the response prolongation during recordings (see Discussion). However, the result strongly suggests that both cAMP and Ca 2+ are present in cilia for such a long time.
When [Ca 2+ ] o was reduced, the half decay time was significantly prolonged, even immediately after the establishment of the WC recording configuration (3 min in Fig. 2 D; white circles). It has been reported that odor responses are prolonged in time course when the external Ca 2+ is removed (Kurahashi, 1989) or when the membrane is held at positive potentials in which Ca 2+ influx is reduced Kurahashi, 2002, 2003). One of the obvious reasons for the extension was that the adaptation was lowered in these conditions. The short-term adaptation in the olfactory cilia is regulated by the influx of Ca 2+ that passes through the CNG channel (Kurahashi, 1990;Kurahashi and Shibuya, 1990;Chen and Yau, 1994;Balasubramanian et al., 1996;Kurahashi and Menini, 1997;Leinders-Zufall et al., 1997;Kleene, 1999;Bradley et al., 2001Bradley et al., , 2004Munger et al., 2001;Boccaccio et al., 2006;Stephan et al., 2011;De Palo et al., 2012;Li et al., 2016). This adaptation is almost completely abolished when the Ca 2+ feedback to the transduction machinery stops (Kurahashi, 1990;Kurahashi and Shibuya, 1990).
In addition, the response was prolonged gradually during the recording even in the 0-added Ca 2+ media (Fig. 2 D, white circles). Such a time-dependent prolongation cannot be explained by the reduction of Ca 2+ -dependent adaptation. The results again suggest that cAMP and Ca 2+ stay in cilia longer than free diffusional processes (see also below). Similarly to this study, experiments using the suction pipette recording that does not affect cytoplasmic factors have shown that the odorant response lasts for >5 s in the absence of external Na + (in these conditions, Ca 2+ extrusion by Na + /Ca 2+ exchange is reduced; Reisert and Matthews, 2001). The long-lasting response could presumably be due to the continuous activation of the Cl − channel by cytoplasmic Ca 2+ . Therefore, it seems likely that Ca 2+ stays within cilia for a long time even when cytoplasmic factors are not manipulated.
Double-pulse stimuli at two tiny areas within the single cilium In a previous work (Takeuchi and Kurahashi, 2008), we applied double-pulse stimuli at two distinct points within a single olfactory cilium. The second response induced at the second region was not affected by the presence of a preceding stimulus at the first region, which was applied a few micrometers away from the second region. This suggested that second messenger molecules do not travel such a distance. However, the result could even be explained when the cAMP-mediated response summation and Ca 2+ -dependent adaptation canceled each other. To discriminate between two possibilities, in this study, we stimulated two different tiny areas successively with varied distances (for methods, see Fig. 1).
A certain part of a single cilium (or two close areas) was selected with an ROI and stimulated twice successively with the photolysis of caged cAMP (Fig. 3 A). We observed two opposite effects in the secondary responses; increments (Fig. 3, A-F) and reductions (Fig. 4, A-F). Response increments were considered the nonlinear summation of responses caused by the presence of basal cAMP as documented in detail in the past (Lowe and Gold, 1993;Kurahashi, 2003, 2008). In contrast, the response reductions represented Ca 2+ -dependent adaptation (Kurahashi and Menini, 1997;Takeuchi and Kurahashi, 2003), indicating that Ca 2+ travels to the secondary-stimulated region. Again, it is important to note that the adaptation is almost completely abolished upon omission of the Ca 2+ feedback (Kurahashi, 1990;Kurahashi and Shibuya, 1990).
We systematically confirmed the appearance of either summation or adaptation by monitoring the change in the secondary response when the stimulus strength for the first region was varied (Fig. 3). We interpreted the response as the summation of [cAMP] i , when the secondary response increased as the first response increased (Fig. 3, E and F) and as adaptation when the secondary response reduced as the first response increased (Fig. 4, A-F). Of 12 ORCs, six showed summation and three showed adaptation, whereas three ORCs showed neither. The three ORCs that showed neither summation nor adaptation may indicate the absence of the function and/or spreading of second messengers. At the same time, it is possible that in these cells, adaptation and summation may have compensated for each other. These matters were not analyzed further in this study.
It was puzzling to see that depending on the sample, either of these two phenomena became dominant. Presumably, this represented the complex mixture of several factors that include differences in cell conditions (basal concentration of second messenger molecules), double-pulse interval, distance of ROIs, variation of buffering and extrusion/degradation systems, and other parameters. At this moment, we are unable to describe their causes systematically. After the identification of summation or adaptation, we changed the distance between two ROIs. Because the summation was caused by cAMP and adaptation by Ca 2+ , such experiments can investigate the cAMP and Ca 2+ spreading (Fig. 5, A-I). As positions of ROIs were changed, the summation and adaptive effects were reduced and became undetectable at ∼2-3 µm (Fig. 5 J). The result indicated that spreading of cAMP and Ca 2+ that are effective in physiological functions in the cilium is restricted within this distance.
So far, molecular spreading in a single cilium was investigated within relatively small time window (e.g., <10 min) after the establishment of the WC recording configuration. Under such conditions, apparent spreading was found to be limited within 2-3-µm distance (Fig. 5 J). We also checked whether such limitation was kept for a long time during the recording because in a longer time window, soluble factors are assumed to be washed out from the cytoplasm. Especially, we examined the relation between a change in the current shape and the distance between two ROIs and the time after the rupture of patch membrane (Fig. 6 A). The current ratio was obtained as the ratio between the second response after the first stimulus and second response only (Fig. 6, B-E). We did not observe any cells in which the second response reduced with the presence of the first response. Therefore, it seemed likely that the adaptation was abolished in such conditions (Fig. 6 F) Fig. 6 F), cAMP spreading that was estimated from the summation of responses was again reduced at distance >2 µm, even after 25 min. Interestingly, however, we could observe in a cell (pink bar) that cAMP spreading was recognized as a response summation even at two stimulating points >2 µm, which was not observed at the early time window during the WC recording. The results suggested that soluble factors (Ca-CAM, Song et al., 2008, and/or phosphodiesterase [PDE]) are, at least in part, involved in the limitation of spreading because prolongation of the cAMP-induced response can at least be explained by the washout (and/or dysfunction) of such factors. Data from E. In this cell, the response induced at a proximal part (second in A) was smaller than that induced at a distal position (first in A), which is unusual because the response is generally bigger at the proximal part. There is a possibility that the edge of the first stimulus also stimulated the neighboring cilium. Ca 2+ diffusion in a fine glass capillary As a control experiment, we measured Ca 2+ diffusion in a thin glass capillary with ∼1-µm inner diameter. The capillary was filled with caged Ca and Fluo-4, and a local area of ∼1-µm diameter was illuminated by a UV laser (Fig. 7 A, arrow). The Ca 2+ signal increased immediately after irradiation, after which it decreased rapidly at the stimulus center and spread laterally along the capillary (Fig. 7 B). The fluorescence distribution could be fitted by the Gaussian function (Fig. 8, A and B). By plotting σ 2 against time (Fig. 8 C), we obtained the diffusion coefficient (D) of 440 ± 35 µm 2 /s (n = 11), which was in good agreement with standard D Ca 2+ (600 µm 2 /s; Hodgkin and Keynes, 1957;Blaustein and Hodgkin, 1969). In 1.8 ± 1.0 s (n = 5), the fluorescence became almost undetectable, which was much faster than WC responses, especially at long-lasting WC recording, or than the time needed for recovery from the adaptation. Therefore, we again recognized that the olfactory cilia have functions to extend the lifetimes of cAMP and Ca 2+ effects.

. In two cells (violet and indigo bars in
Digital model for the random walk in the presence of binding sites Longitudinal spreading of second messenger molecules in the olfactory cilia is highly likely to be strongly restricted, at least in physiologically reliable concentration ranges. There are several possibilities to explain how the spreading is limited  in the smooth tubular structure. Cytoplasmic buffers and extrusion and cAMP and Ca 2+ degradation will be at least in part responsible for the reduction of spreading of these molecules during their diffusion. In a previous work, however, Takeuchi and Kurahashi (2008) showed that the degree of adaptation and recovery time course with a stimulus onto a confined region did not change remarkably from those observed upon the entire-cilia stimulation (Kurahashi and Menini, 1997). It is puzzling why the cAMP response and Ca 2+ -dependent adaptation stay for >10 s even with a very confined stimulation; these molecules should quickly disappear if the rapid extrusion/degradation and buffers were the entire mechanism for restricted spreading.
We speculated that the anomalously thin structure of cilia may be responsible for the limitation of diffusional processes, mostly because of the high surface/volume ratio. In the same concentration of the solution in a cylindrical space, the binding site at the cytoplasmic surface of the plasma membrane becomes dominant to affect diffusion. To verify this possibility, we constructed a digital model that actually simulates the random walk process of molecules in the absence (Fig. 9, A and B) and presence (Fig. 10, A and B) of binding sites on the cytoplasmic surface of the plasma membrane.
When the cytoplasmic surface did not have any binding sites, free molecules spread along the x axis (Fig. 11, A and B) and σ 2 gradually became wider with time (Fig. 11, E and F), showing a linear change in the time process (Fig. 11 I). The diffusion process was not affected by a change in the diameter when the membrane did not have binding sites (Fig. 11 J). When the membrane had binding sites that held the molecule, for example 1.16 µs, D was strongly reduced (Fig. 11, C, D, G, and H). The relation between σ 2 and stickiness showed nearly an exponential decrease (Fig. 11 M). In contrast with the previous measurements, D strongly depended on the diameter of the process (Fig. 11, K and L). Plotting σ 2 against the diameter, it became obvious that diffusion actually depended on the diameter of the process when the cytoplasmic surface of the membrane had binding sites (Fig. 11 N). It is highly likely that the diffusion of soluble molecules in an anomalously fine tube like the cilium is dramatically reduced on the basis of the diameter when the cytoplasmic surface of the membrane has binding sites.

Discussion
This study showed that both cAMP and Ca 2+ work only in the vicinity of the site of generation in the olfactory cilia. The result was consistent with the fact that ORCs show an integer multiple of unitary events observed at low Ca 2+ conditions (Bhandawat et al., 2005), which does not occur when second messengers spread rapidly over the cilium. A similar compartmentalization has been shown in photoreceptor cells in which Ca 2+ independently works between the outer and inner segments that are connected via a thin ciliary structure (connecting cilium; Krizaj and Copenhagen, 1998;Nair et al., 2005;Calvert et al., 2010). Some of our present findings may be related to the presence of a microdomain in the olfactory cilia (Castillo et al., 2010;Pietrobon et al., 2011;López et al., 2014;Henkel et al., 2015). In the primary cilia, Ca 2+ signaling is also uncoupled from signaling in the cell cytoplasm (Delling et al., 2013). These findings provide examples of functional polarity in compact cells. It seems likely that the cytoplasmic buffers, extrusion systems, and degradation systems are involved in this limitation of the spreading of molecules. Additionally, it is possible that  the diffusion process strongly slows down in such a narrow space because of binding sites on the cytoplasmic surface of the plasma membrane as shown in the digital model. These properties may enable ORCs to express a long-lasting response and adaptation. The lifetime of the cAMP-induced response and recovery from adaptation were much longer than the time course for the disappearance of Ca 2+ signals within the glass capillary. Interestingly, Ca 2+ in the dendrosomatic region does not backpropagate into the cilia, either. The response kinetics of the odorant-induced current is unaffected by the step depolarization under the voltage clamp condition that activates voltage-gated Ca 2+ channels located in the dendrosomatic membrane (see e.g., Kurahashi, 1989). It is also shown that Ca 2+ signals measured with Fluo3 in the cilia did not show any sign for backpropagation of Ca 2+ from the knob when a cilium was stimulated by an odor or the cell body was stimulated by a high K solution (Leinders-Zufall et al., 1997. In this study, we found that the time courses of cAMP-and Ca 2+ -induced currents prolonged during WC recordings. At this moment, the precise mechanism for this phenomenon is unknown. It is unlikely that the long-lasting WC recording configuration inhibits cAMP and Ca 2+ diffusion in cilia; conversely, a reduction in the activities of several factors may be involved in the prolonged time course. For instance, PDE activity is one possibility; the cAMP-dependent response is expected to be prolonged when PDE activities are lowered. Ca-CAM (Song et al., 2008) is another candidate. Na + /Ca 2+ exchange is also reportedly involved in the response termination (Stephan et al., 2011). Furthermore, the prolongation of the Ca 2+ response (Fig. 2 E) may be caused by a reduction of [ATP] i that is involved in Ca 2+ extrusion (Antolin et al., 2010). Chen et al. (1999) showed that the cAMP response of a single olfactory cilium drops within a few seconds after the washout of cytoplasmic cAMP (Chen et al., 1999). For their recording, they used a detached single olfactory cilium preparation to load and wash out cAMP from the proximal part of the cilium. In contrast, in this study, we introduced cAMP from the WC pipette situated at the dendrosomatic area. When cAMP concentration was jumped locally by photolysis of caged cAMP at the middle part of the cilium, the lifetime of the cAMP response was sometimes >10 s. There are several differences between these experimental conditions. One of the biggest differences could be the concentration of cAMP. They used [cAMP] i <20 µM, whereas in this study, [cAMP] i could be higher at the place of irradiation (ROI); we have estimated that ciliary [cAMP] i could be as high as 100 µM when the cell was stimulated by odorants or caged cAMP (Takeuchi and Kurahashi, 2005). When [cAMP] i becomes high, buffering effects become dominant (Chen et al., 1999), which would slow down the diffusional processes. Moreover, cAMP and/or Ca 2+ diffusion, the washout of cytoplasmic factors, or special distribution of voltage profiles along the cilium may have contributed to the variance. Further experiments are warranted to clarify this.
It is controversial whether cAMP that is generated in cilia upon odorant stimulation diffuses to the dendrosomatic regions (e.g., Reisert and Zhao, 2011). cAMP plays important roles in the cell body for (a) the change in the spiking properties via an A-kinase (Kawai et al., 1999), and (b) the determination of basal activity via an HCN channel (Nakashima et al., 2013) that may be related to selective axonal projection (Imai et al., 2006;Imai and Sakano, 2009;Mobley et al., 2010). Our finding regarding slow diffusion and well-known degradation in the olfactory cilia indicates that cAMP generated in the cilia does not affect cellular activities conducted at the cell body. The geometrical spatial ratio between the cilia and the dendrosomatic regions needs to be considered. The total volume of the cilia is 1 πμm 3 (0.2 µm diameter and 10 µm length; 10 cilia), whereas that of dendrosomatic regions is 470 πμm 3 (dendrite, 1 µm diameter and 50 µm length; cell body, 14 µm diameter sphere), providing a ratio of ∼500. Even if there was no diffusion limitation or extrusion/degradation in cilia, 10 µM cAMP generated at cilia became 20 nM in the dendrosomatic regions (Takeuchi and Kurahashi, 2005), which is already lower than the physiologically reliable range.
This study showed that the diffusion of molecules slows down based on diameter of processes when the cytoplasmic surface of the plasma membrane has binding sites. The cytoplasmic side of the olfactory cilia has several binding sites for cAMP and Ca 2+ (e.g., microdomain in cilia, Castillo et al., 2010). The ciliary membrane contains an anomalously high density of CNG channels Kaneko, 1991, 1993), 700-2,300 molecules of CNG channels in 1-µm length of the olfactory cilium (Kurahashi, 1990). Each CNG channel forms a tetramer, having four binding sites in individual molecules. Therefore, the binding sites for cAMP molecules are 2,800-9,200 per 1-µm length of the cilium. The cAMP concentration reaches as high as 100 µM at the maximum (Takeuchi and Kurahashi, 2005), which gives 450 molecules per 1-µm length of the cilium, much smaller than the binding capacity for cAMP. With regard to the binding sites for Ca 2+ , CaM is permanently bound to the cytoplasmic domain of each CNG channel (Chen and Yau, 1994;Bradley et al., 2001Bradley et al., , 2004. In addition, lipid bilayers and various proteins including tubulin may serve as strong binding sites for Ca 2+ . Besides, unidentified Ca 2+ -binding proteins (Balasubramanian et al., 1996) and Cl (Ca) channels (Kleene and Gesteland, 1991;Kurahashi and Yau, 1993;Reisert and Matthews, 1998) are well-known Ca 2+ -binding sites. All of these binding sites may be responsible for limiting diffusion. Simultaneously, cAMP is degraded by PDE (Cygnar and Zhao, 2009), whereas Ca 2+ is extruded by Na + /Ca 2+ exchanger (Castillo et al., 2007;Stephan et al., 2011) and ATP-dependent Ca 2+ extrusion (Antolin et al., 2010), which is involved in the restriction of molecular spreading. For a long time, it was believed that the ciliary structure for sensory perception is responsible for the increase in the probability of stimulant capture. As the molecular dynamics were revealed, it became clear that this fine structure is quite beneficial for efficient signal transduction. As described in this study, the narrow space can limit cytoplasmic molecule diffusions, and as a result, the response becomes prolonged and adaptation lasts for a long time. The high surface/volume ratio is responsible for a rapid increase in the molecular concentration even with a low production and with a small influx of molecules. Altogether, signal transduction is achieved efficiently in such a narrow space. Such efficient energy conversion may also be broadly used in other biological systems, which have not yet been subjected to systematic experiments. Note that σ 2 is dependent on the diameter.


s2fieldsofstudy: Biology Medicine

text:
The Tumor Microenvironment of Epithelial Ovarian Cancer and Its Influence on Response to Immunotherapy

Immunotherapy as a treatment for cancer is a growing field of endeavor but reports of success have been limited for epithelial ovarian cancer. Overcoming the challenges to developing more effective therapeutic approaches lies in a better understanding of the factors in cancer cells and the surrounding tumor microenvironment that limit response to immunotherapies. This article provides an overview of some ovarian cancer cell features such as tumor-associated antigens, ovarian cancer-derived exosomes, tumor mutational burden and overexpression of immunoinhibitory molecules. Moreover, we describe relevant cell types found in epithelial ovarian tumors including immune cells (T and B lymphocytes, Tregs, NK cells, TAMs, MDSCs) and other components found in the tumor microenvironment including fibroblasts and the adipocytes in the omentum. We focus on how those components may influence responses to standard treatments or immunotherapies.

Introduction
An increasing body of evidence strongly suggests that the immune system is able to identify, control and eliminate nascent neoplastic cells in a process known as cancer immunosurveillance [1]. Epithelial ovarian cancers (EOCs) are "immunogenic tumors" that produce spontaneous antitumor immune responses detectable in peripheral blood, tumors and ascites of patients [2][3][4]. The resulting presence of tumor infiltrating lymphocytes (TILs) is associated with improved survival in EOC [5]. Unfortunately, there are a number of factors in the tumor microenvironment (TME) that can impair the presence or activity of TILs, thereby facilitating cancer progression.
Various immunotherapeutic strategies are attempting to address the challenges posed by the highly immunosuppressive EOC TME. Immunotherapies encompass many modalities, including immune checkpoint blockade, antibody-based therapies, cancer vaccines, cytokines, adoptive cell transfer, and chimeric antigen receptor-modified T cells [6]. However, emerging cancer immunotherapies (blocking antibodies for checkpoint inhibitors) have shown low rates of responses in EOC (reviewed in [2]). Improving this response rate is a major goal, which can only be achieved with a better understanding of the elements in the TME that contribute to treatment failure. Immune cells are the main players in the development of antitumor immunity or tumor progression, but there are also other components in the TME that should be taken into consideration when designing new therapeutic strategies. Those components include EOC-derived exosomes, cancer-associated fibroblasts (CAFs) and adipocytes residing in the omentum.
In this review we will describe those elements of the TME, how they influence the burden of the tumor, the responses to therapies, and their relevance in designing cancer immunotherapies for EOC.

Cancer Cells and Tumor Antigens
The success of cancer immunotherapy hinges on the ability to generate cancer-specific antitumor T-cell responses, to both recognize tumor-associated antigens (TAAs) and kill tumor cells, and to generate memory responses. TAAs can be classified into different categories: tissue differentiation, cancer testes antigens (CTAs), neoantigens derived from mutations, overexpressed cellular, splice variant, glycolipid, and viral antigens [7,8]. Ideal TAAs for immunotherapy targets are immunogenic and are expressed or overexpressed in tumor tissue, with restricted expression in associated normal tissues, in a significant percentage of patients [9]. Positive responses to immunotherapies such as immune checkpoint inhibitors [blocking programmed cell death protein 1 (PD-l), programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)], have been associated with high mutation/neoantigen burden [10,11]. The initial clinical studies of small numbers of EOC patients treated with immune checkpoint inhibitors have resulted in clinical benefits in less than 20% of patients (Table 1). Unfortunately, little is known about the TME at the start of treatment in most studies, making it impossible to discern the factors that may have blocked any response. The failure to respond could be related to the neoantigen burden in EOC, which may be insufficient to generate a significant antitumoral response [12,13]. There are currently intense research efforts to understand other TAAs (Table 2) recognized by TILs to design informed immunotherapy targets (Table 3). Expression of PD-L1 in ovarian cancer tissues was not significantly correlated with objective response but 16/20 patients having a high expression of PDL1 on tumors did not respond to treatment (vs. 2/4 responders in the PD-L1-low expression group).
Neoantigen depletion [63], intratumoral heterogeneity, and clonal evolution of primary tumors and metastases may influence immunosurveillance and response to immunotherapy [64,65]. Epithelial T-cell rich tumors show the lowest amount of clonal diversity, neoantigen diversity and greatest loss of human leukocyte antigen (HLA) expression, which suggests immunoediting in the TME. T-cell poor tumors or "cold tumors" have a higher predicted and more diverse neoantigen load (unedited) [63].

Cancer Testes Antigens
CTAs are encoded by~140 genes that are normally only expressed in germ cells (testes, placenta, fetal ovary) and not normal somatic adult cells, but often highly expressed in tumors. This along with their immunogenicity makes them significant targets for cancer immunotherapy [9,66,67]. Vaccination with recombinant MAGE-A3 antigen has been used in Phase I/II clinical trials for melanoma [68] and non-small-cell lung cancer (NSCLC) [69] with a good safety profile and observed humoral response, but only slight effects on survival.
Many characteristics of CTA epitopes and all TAAs such as (i) immunogenicity; (ii) restriction to HLA-I or -II; (iii) natural processing; (iv) expression; and (v) role in tumor progression remain to be elucidated and require validation in larger sample sizes. While the expression of CTAs does not often correlate with improved survival, their tissue specificity makes CTAs attractive targets for immunotherapies (Table 3) such as peptide vaccines [44,70], antigen-loaded dendritic cell (DC) vaccines [72], or oncolytic viral platforms, and for combined interventions with immune checkpoint inhibitors [73] or chemotherapy [74], in order to overcome tumor escape mechanisms.

Other TAAs
Genetic and epigenetic aberrations in cancer cells, resulting from mutations, amplifications or deletions in genes, provide both therapeutic targets and potential TAAs for immunotherapy design (Table 2). However, the greatest hurdles still remain in designing immunotherapeutic targets for a disease in which such aberrations, with the exception of p53 mutation (95% of HGSC [33,75]), are relatively uncommon (<20% frequency in HGSC cases) and lack antigen specificity to the tumor. Immunogenic oncogenes p53, Her2-neu and WT1 are broadly overexpressed in EOC, particularly HGSC, and targeted immunotherapies have been explored in clinical trials (Table 3). Other common but infrequent amplifications, mutations or deletions occur in CCNE1, NF1, PTEN, KRAS, RB, CDK2NA, PIK3CA and AKT1/2 and provide potential therapeutic targets for EOC immunotherapy [33]. The DCs, T-cells, and peptide-based vaccine strategies against proteins described above have largely demonstrated immunological responses including CD4+ and CD8+ T-cell responses in preliminary clinical trials following vaccination, but often in the absence of clinical responses. This is perhaps due to widespread immunosuppression in the TME preventing T-cell activation and proliferation, as well as tumor heterogeneity and immunogenicity that impede proper TAA presentation to the immune cells.
The EOC immunopeptidome was profiled by isolating HLA molecules primarily from HGSC tumors and which were analyzed by mass spectrometry [57]. The analysis identified relevant proteins including CRABP1/2, FOLR1, and KLK10 presented on major histocompatibility complex (MHC) I molecules, and mesothelin, PTPRS and UBB presented on MHC-II molecules [57]. The most abundantly detected protein presented on MHC-I molecules was MUC16 (CA-125), with 113 different peptides expressed in approximately 80% of patients. MUC16-derived peptides were highly immunogenic (85% T-cell responses in vitro), and consequently it was proposed as the top candidate for targeted immunotherapy moving forward [57]. Although CA-125 is immunogenic, the large number of trials with a monoclonal antibody targeting CA-125 (Table 3) have been mostly unsuccessful as a monotherapy [76]. This failure could be explained by the weak magnitude of the immune response generated, the loss of expression or down-regulation of CA-125 on EOC cells to avoid immune recognition, or the overgrowth of CA-125(-) EOC cells as a consequence of cancer immunoediting process.
A single TAA is generally only expressed in a subset of patients, making the design of a universal immunotherapy challenging. The main barrier of targeting a single TAA is cancer immunoediting, which enables the enrichment of neoplastic cells in tumors that do not express the targeted TAA over time. Chimeric antigen receptor T (CAR-T) cells provides the option of combining multiple antigen specificities, and delivering direct cytokine stimulation (GM-CSF, IL-12) to the TME, irrespective of the MHC status of the patient [8].

Tumor Immunogenicity and Other Immunoinhibitory Molecules
Loss of immunogenicity is an immune hallmark of cancer that is exploited by tumors to evade immune recognition. This can be triggered by down-regulation or loss of expression of MHC-I and -II, and the antigen processing and presentation machinery (APM) [77][78][79][80]. Expression of MHC-I genes is altered by 60-90%, depending on the cancer type. These impairments reduce the antigens presented on the cell surface leading to decreased or lack of recognition and elimination by cytotoxic T lymphocytes.
The mechanisms that are related to immune cell infiltration in EOC are dependent on MHC-I and -II status [3,81]. The presence of neoantigen-reactive T cells in patients with EOC can improve survival [82]. However, as mentioned before, since ovarian tumors possess intermediate/low mutation burdens, the incidence of naturally processed and presented neoantigens generating a significant antitumoral response is very low [13]. The expression of APM components and the presence of intratumoral T-cell infiltrates were significantly associated with improved survival [81].
Han. et al. demonstrated that the majority of ovarian carcinomas analyzed had either heterogeneous or positive expression of peptide transporter 1 (TAP1), TAP2, HLA class I heavy chain, and beta-2 microglobulin [81]. Concurrent expression of HLA-DR and CA-125 on cancer cells correlated with higher frequency of CD8+ TILs and increased survival [83]. Similarly, tumor cell expression of HLA-DMB was associated with increased numbers of CD8+ TILs and both were associated with improved survival in advanced-stage serous EOC [84]. The regulation of APM components and MHC molecules in human cancers is a significant area of research but is beyond the scope of this review (reviewed in [85,86]).
The mutational profile of EOC can also predict immunogenicity. Tumors with deficient homologous recombination (HR) machinery occur with a frequency of up to 50% [33]. These include mutations in BRCA1/BRCA2 (20% frequency) or non-BRCA HR deficiencies (Fanconi anemia genes, restriction site associated DNA genes, and DNA damage response genes) [33]. HR deficient tumors have higher predicted neoantigen load, and infiltrating and peritumoral lymphocytes in these tumors have increased PD-1/PD-L1 expression [43], which may enhance susceptibility to immune checkpoint therapy. BRCA1/2 mutated HGSC tumors have more CD3+ and CD8+ TILs compared to HR-proficient tumors, a signature associated with higher overall survival [43,87]. p53 mutations are also associated with higher levels of TILs [87,88]. Non-HR deficient tumors therefore have poorer overall survival [43] and may be less immunogenic, making them more difficult to target with immunotherapies. Alternative strategies and TAAs to target this group of EOC tumors need further investigation.
The expression of immunoinhibitory molecules on cancer cells, including PD-L1 and Indoleamine 2,3-dioxygenase (IDO) are associated with patient prognosis. Higher expression of PD-L1 on tumor cells correlates with poorer prognosis, suggesting that the PD-1/PD-L pathway can be a good target for restoring antitumor immunity in EOC [89,90], although others have suggested that high PD-1/PD-L1 expression in primary tumors may be associated with a favorable progression-free survival [91,92]. Increased infiltration of CD8+ T cells is associated with high PD-L1 expression likely as a result of an adaptive response where infiltrating CD8+ T cells secrete interferon gamma (IFNγ) that subsequently induces PD-L1 expression on cancer cells. This in turn inhibits T-cell activation and proliferation, preventing successful targeting and clearance of the tumor. Immune checkpoint inhibitors (anti-PD-L1 and PD-1) have been FDA approved for melanoma and NSCLC, but only a small percentage (10-33%) of ovarian cancers express PD-L1 [61,92,93], thus only a small percentage of patients may respond to anti-PD-L1 immunotherapy ( Table 1). The enzyme IDO is often overexpressed by cancer cells, but is also produced by DCs and macrophages [94,95] in the TME. IDO catabolizes tryptophan, which leads to cell cycle arrest or apoptosis in NK and CD4 T cells [96], and skewed differentiation of regulatory T cells (Tregs) induced by plasmacytoid DCs, leading to immunosuppression in the TME [97]. Positive staining for IDO, observed in 24-57% of patient samples, is associated with poor prognosis of HGSC, decreased CD8+ TILs, as well as resistance to chemotherapy [98,99]. Targeting IDO with inhibitors may improve outcome [100,101].

Immune Cells
Most solid tumors are infiltrated by myeloid-and lymphoid lineage-derived immune cells that are differentially distributed within the TME with a crucial role in the establishment of antitumoral responses or tumor progression [1]. Growing tumor cells release "danger signals" that enable the recruitment of immune cells into the tumor niche. TILs such as CD4+ and CD8+ T cells, B lymphocytes, Natural Killer (NK)-T cells, as well as innate immune cells such as NK cells, macrophages and DCs, are then recruited in order to eliminate nascent neoplastic cells, acting as an extrinsic tumor suppression mechanism [102]. However, immunosurveillance promotes the selection of poorly immunogenic cancer cells through cancer immunoediting where neoplastic cells that resist the elimination phase can persist in equilibrium with effector CD4+ and CD8+ T cells under a pro-inflammatory milieu. Over time, cancer cells with the most immunoevasive characteristics are selected, enabling them to eventually escape immune attack [102]. Finally, immunoedited tumors become clinically apparent with variants that trigger the establishment of an immunosuppressive TME containing immunosuppressive immune cells such as myeloid-derived suppressor cells (MDSCs), Tregs, and others [2,103].

TILs
TILs can localize into the tumor islet (intraepithelial) and in the peritumoral space (stromal) [2]. Several studies have shown a positive correlation between the presence of intraepithelial TILs and tumor regression in many solid cancers [4,5,[104][105][106][107]. T cells can be found in primary tumor tissue and omental metastases [4,104,105,[107][108][109][110][111] and their presence has been correlated with positive prognosis. Dadmarz et al. demonstrated that TILs isolated from EOC patients (primary tumor, metastases or ascites) were tumor-specific and could recognize autologous TAAs. Antitumoral responses were mainly characterized by the secretion of tumor necrosis factor-alpha (TNFα) and granulocyte macrophage-colony stimulating factor (GM-CSF) when stimulated with autologous tumor [112]. Later, Zhang and colleagues showed that intraepithelial CD3+ TILs can be found in >50% of advanced-stage EOC with their presence correlating with a five-year overall survival rate of 38% in contrast to 4.5% in patients whose tumors contained no T cells [5]. Even after debulking and platinum-based chemotherapy, the presence of intraepithelial CD3+ TILs increased the five-year overall survival rate (>70%) in comparison to patients whose tumors contained no T cells in islets (11%) [5]. T cell-rich tumors correlated with delayed recurrence or death and were associated with increased expression of Interleukin-2 (IL-2), IFNγ and lymphocyte-attracting chemokines within the tumor such as CXCL9 [113], CCL21, and CCL22 [5]. Conversely, tumors with no T cells in islets were associated with an increased level of vascular endothelial growth factor (VEGF), an angiogenic regulatory factor in the TME associated with early recurrence and short survival [5]. A more recent study showed that intratumoral accumulation of CXCR3 ligands such as CXCL9 and CXCL10, predicts survival in advanced HGSC [113] (Figure 1). This study also identified the cyclooxygenase (COX) metabolite Prostaglandin E2 as a negative regulator of chemokine secretion that contributes to tumor progression by impeding TILs recruitment in ovarian cancer [113]. Further investigation showed that expression of both COX-1 and COX-2 were negatively correlated with intraepithelial CD8+ TILs as well as with EOC patient survival [114].
In 2012, a meta-analysis of ten studies with 1815 ovarian cancer patients confirmed the prognostic value of intraepithelial CD8+ TILs in EOC specimens regardless of the tumor grade, stage, or histologic subtype studied [111]. Their presence suggests that spontaneously activated antitumoral responses are present in the tumor niche to control tumor outgrowth [111] as observed by the presence of tumor-reactive antibodies and T cells found in the peripheral blood of advanced stage EOC patients [116][117][118], and oligoclonal tumor-reactive T cells isolated from blood, ascites or tumors [88,[119][120][121][122][123]. Conversely, the lack of intraepithelial TILs is significantly associated with poor survival among EOC patients [111]. Thus, immunotherapies aiming to increase the effector functions of pre-existing antitumoral CD8+ TILs and triggering effector T cell-trafficking to the TME are the holy grail of cancer immunotherapy.
CD4+ T cells as well as CD8+ T cells can specifically recognize TAAs from malignant cells. CD4+ T helper (Th) cells provide cytokine support for CD8+ T-cell proliferation and expansion to eliminate cancer cells and trigger antitumoral responses. In an analysis of ovarian tumors, Tsiatas et al. found that a high percentage of CD4+ CD25hi cells and activated CD4+ T cells were significantly associated with improved median overall survival [124]. Two other studies also showed a positive correlation of the high frequency of CD4+ TILs and EOC patient survival [110,125]. Nesbeth et al., using an animal model for EOC, found that tumor-primed CD4+ T cells produce high levels of CCL5 that enables the recruitment and activation of DCs to the TME. Mature DCs were then able to prime tumor-specific CD8+ T cells and confer long-term protection [126]. Hence, immunotherapies stimulating both effector CD4+ and CD8+ T cells could confer synergistic antitumoral responses.
Cancers 2018, 10, x 10 of 29 enables the recruitment and activation of DCs to the TME. Mature DCs were then able to prime tumor-specific CD8+ T cells and confer long-term protection [126]. Hence, immunotherapies stimulating both effector CD4+ and CD8+ T cells could confer synergistic antitumoral responses.

Regulatory T lymphocytes
Tregs negatively regulate antitumoral responses in both a direct and indirect manner, highlighting that Tregs are a fundamental means of tumor immune evasion [127,128]. In healthy tissues, Tregs mediate tolerance by suppressing autoreactive T cells to protect and prevent excessive tissue destruction. Since most TAAs are composed by self-peptides, Tregs are often found in tumors to dampen antitumoral responses. Tregs accumulate and are more frequently present in tumors, with a shift in the median ratio of Tregs to TILs from 3-8% in healthy tissue to 18-25% in all analyzed cancers, including EOC [129]. Curiel et al. analyzed 104 EOC specimens and found that CD4+ CD25+ FOXP3+ Tregs specifically suppress antitumoral T cells in vivo, contributing to tumor growth. In addition, their presence correlates with poor patient outcome [130]. CD4+ Tregs preferentially migrated to tumor and ascites and were rarely found in draining lymph nodes at later cancer stages [131]. Immunotherapies impeding Treg trafficking could release the TME immunosuppression and promote the development of antitumoral responses.

Regulatory T lymphocytes
Tregs negatively regulate antitumoral responses in both a direct and indirect manner, highlighting that Tregs are a fundamental means of tumor immune evasion [127,128]. In healthy tissues, Tregs mediate tolerance by suppressing autoreactive T cells to protect and prevent excessive tissue destruction. Since most TAAs are composed by self-peptides, Tregs are often found in tumors to dampen antitumoral responses. Tregs accumulate and are more frequently present in tumors, with a shift in the median ratio of Tregs to TILs from 3-8% in healthy tissue to 18-25% in all analyzed cancers, including EOC [129]. Curiel et al. analyzed 104 EOC specimens and found that CD4+ CD25+ FOXP3+ Tregs specifically suppress antitumoral T cells in vivo, contributing to tumor growth. In addition, their presence correlates with poor patient outcome [130]. CD4+ Tregs preferentially migrated to tumor and ascites and were rarely found in draining lymph nodes at later cancer stages [131]. Immunotherapies impeding Treg trafficking could release the TME immunosuppression and promote the development of antitumoral responses. FOXP3+ Tregs express minimal levels of effector cytokines and granzyme B, but are able to induce inhibitory activities through IL-10 and transforming growth factor beta (TGF-β) production [132] and cell-cell interactions [127] (Figure 2). Barnett et al. showed that EOC tumors highly infiltrated by Tregs were associated with poor survival, advanced stage and suboptimal debulking [109]. Investigation of the influence of cytoreduction on the immune system of primary and recurrent EOC found that the ratio of CD4/CD8 is increased in primary but not in recurrent tumors [133]. Primary cytoreduction increased circulating effector CD4+ and CD8+ T cells, but circulating CD4+ Tregs were decreased as well as IL-10 serum levels, but not TGFβ and IL-6 [133]. CD4+ Tregs were also decreased after chemical debulking in patients treated with neoadjuvant chemotherapy. The reduction of the systemic and TME immunosuppression triggered by surgical debulking resulted in an increased capacity of CD8+ T cells to respond to the recall antigens, but not in patients who were previously subjected to chemotherapy or affected by recurrent EOC [133].
Fialová and colleagues studied the dynamics of the tumor-infiltrating immune cells during different stages of EOC [134]. Early stage disease displayed a strong Th17 immune response while stage II patients had responses characterized by the recruitment of Th1 cells. Disseminated disease (stages III and IV) were characterized by high amounts of Tregs, tumor-associated macrophages (TAMs), DCs, and high levels of CCL22, which is secreted by tumor cells, TAMs and DCs to enable further recruitment of Tregs and immunosuppression [134]. Other studies have shown the importance of the TME in facilitating the establishment of tolerance and recruitment of Tregs to sustain tumor growth. Using EOC cell lines in vitro, Facciabene et al. found that tumor hypoxia induces the expression of chemokine ligands such as CCL28, enabling the recruitment of Tregs and triggering angiogenesis [135]. CCL28 overexpression was associated with a poor outcome in patients with EOC [135]. Similarly, CCL22 production by TAMs enabled the recruitment of Tregs [130,134] that induced B7-H4 on antigen-presenting cells including macrophages [136]. CXCR3+ Tregs, able to control type-I T-cell responses, are highly enriched in EOC and represent the majority of Tregs [137]. These Tregs were able to suppress T-cell proliferation and IFNγ secretion [137].
An interesting study analyzed 22 EOC ascites specimens and found significantly elevated levels of IL-6, IL-8, IL-10, IL-15, IP-10, MCP-1, MIP-1β and VEGF and significantly reduced levels of IL-2, IL-5, IL-7, IL-17, PDGF-BB, and CCL5 compared to plasma. Moreover, T cells derived from EOC-associated ascites displayed poor responsiveness when expanded in vitro [138]. The authors claimed that this non-responsiveness could be explained by a high CD4/CD8 ratio that may indicate the presence of Tregs, reduced IL-2 and elevated IL-6 and IL-10 levels triggering a Th2 inhibitory immune response [138]. This high CD4/CD8 ratio was also associated with poor outcome [109,115,136,139], consistent with other studies [124,140,141]. In contrast, a positive correlation between Tregs and patient prognosis has been reported [140]. High tumor grade correlated with higher frequencies of CD3+, CD68+ CD163+ TAMs, and CD25+ FOXP3+ Treg cells, but Treg frequencies were significant predictors of favorable prognosis in patients with familial ovarian cancer (11/73 patients with BRCA mutation) [140]. The presence of FOXP3+ TILs may be linked to positive prognostic factors in optimally debulked HGSC patients [141]. Nevertheless, this disease-specific survival was positively associated along with other TIL markers such as CD8, CD3, TIA-1, CD20 (a B cell surface marker), MHC class I and class II [141]. CD8+ Tregs are also found in EOC [142,143]. They regulate the immunosuppressive TME by limiting immunosurveillance mechanisms and contributing to cancer progression [144]. Recently, Zhang and colleagues showed that CD8+ Tregs are found in the stroma and intraepithelial areas of EOC tumors [143]. CD8+ Tregs are characterized by the expression of FOXP3, CTLA-4, and CD25, but decreased expression of CD28 [143]. CD8+ Tregs were able to convert effector CD8+ T cells into suppressor cells [143]. CD8+ Tregs exert their suppressive function through the secretion of TGF-β1 [142].
Overall, Tregs are considered a critical barrier against antitumoral responses along with tolerance-inducing plasmacytoid DCs, B7-H4+ TAMs, MDSCs, IL-10, TGFβ, and VEGF. All these processes act in concert as a tumor evasion mechanism resulting in tumor progression [145]. Barriers to antitumoral responses are summarized in Figure 2. Tumor promoting network in the EOC TME. Outgrowth of EOC provokes hypoxia that induces the expression of chemokines to recruit MDSCs, Tregs, and TAMs. Tregs induce B7-H4 expression on APCs, subsequently blocking cytokine secretion, cytolytic activity, T-cell proliferation and promoting an immunosuppressive TME. EOC cells and MDSCs produce IDO that catabolizes tryptophan, rendering T cells anergic and dysfunctional. MDSCs and TAMs contribute to tumor growth, malignancy, metastasis and stemness. Several tumor promoting cytokines such as IL-6, IL-10 and TGFβ are prominent in the TME. VEGF released by EOC cells and CAFs stimulates angiogenic factors in the TME. CAFs also secrete many factors that mediate tumor cell migration, proliferation, invasion and chemoresistance, and contribute to the immunosuppressive TME. Adipocytes produce FA and cytokines that fuel tumor growth and omental metastasis. Myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), regulatory T cells (Tregs), cancer-associated fibroblasts (CAFs), fatty acids (FA).

B Lymphocytes
B lymphocytes have been reported to have pivotal roles in cancer immunity [146]. Stromal or intraepithelial B lymphocytes have been found in EOC [141]; however their function in tumor development is not yet clear. Their presence is proposed to be associated with a good prognosis depending on the tumor stage and the TME where they are found [4,108,141]. The presence of B cells Figure 2. Tumor promoting network in the EOC TME. Outgrowth of EOC provokes hypoxia that induces the expression of chemokines to recruit MDSCs, Tregs, and TAMs. Tregs induce B7-H4 expression on APCs, subsequently blocking cytokine secretion, cytolytic activity, T-cell proliferation and promoting an immunosuppressive TME. EOC cells and MDSCs produce IDO that catabolizes tryptophan, rendering T cells anergic and dysfunctional. MDSCs and TAMs contribute to tumor growth, malignancy, metastasis and stemness. Several tumor promoting cytokines such as IL-6, IL-10 and TGFβ are prominent in the TME. VEGF released by EOC cells and CAFs stimulates angiogenic factors in the TME. CAFs also secrete many factors that mediate tumor cell migration, proliferation, invasion and chemoresistance, and contribute to the immunosuppressive TME. Adipocytes produce FA and cytokines that fuel tumor growth and omental metastasis. Myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), regulatory T cells (Tregs), cancer-associated fibroblasts (CAFs), fatty acids (FA).

B Lymphocytes
B lymphocytes have been reported to have pivotal roles in cancer immunity [146]. Stromal or intraepithelial B lymphocytes have been found in EOC [141]; however their function in tumor development is not yet clear. Their presence is proposed to be associated with a good prognosis depending on the tumor stage and the TME where they are found [4,108,141]. The presence of B cells and CD8+ TILs correlates with increased patient survival compared to CD8+ TILs alone [108]. Nielsen et al. analyzed tumor and serum specimens obtained from patients with HGSC and found that the majority of CD20+ TILs were antigen experienced and suggested to accomplish TAA presentation in the TME since they often co-localized with CD8+ TILs and expressed markers such as MHC-I, MHC-II, CD40, CD80, and CD86 [108]. B cells can achieve antitumor immunity by secreting IFNγ, facilitating CD4+ Th cells to polarize to Th1 responses, and promote T-cell expansion by presenting TAAs [146]. Recently, the positive role of B cells among TILs at metastatic sites from patients with HGSC was reported [147]. B cells were often found in the stroma of metastases and were characterized by a strong memory response against TAAs by production of tumor-specific IgGs (Figure 1). Interestingly, these responses were amplified by chemotherapy [147].
Conversely, a new subset of B cells, regulatory B cells (Bregs), has been recently designated as immunosuppressive cells able to secrete anti-inflammatory mediators such as IL-10, IL-35, and TGF-β, triggering T-cell conversion to Tregs [148] (Figure 2). Indeed, a study that analyzed EOC tumor tissue and omental metastases found that high B cell infiltration negatively correlates with patient survival [149]. High CD20 and CD138 expression correlated with high tumor grade [149]. Analysis of omental specimens from patients with HGSC found that overall survival was 160.6 months in patients with low B-cell expression vs. 47.3 months in those with high B-cell expression, associating increased B-cell infiltration with poorer survival [150]. Similarly, the analysis of post-chemotherapy effusions from ovarian carcinomas revealed that a higher percentage of CD19+ cells (B cell marker) and stage IV disease predicted poor survival for patients [151].
Taken together, it is important to consider that several B-cell subsets with different phenotypes and functions exist, and they may have various roles in modifying the ability of tumors to respond to treatment [146]. Thus, a deep characterization of B-cell subpopulations within the TILs, ascites, and peripheral blood at different stages is crucial in order to provide a better understanding of the capability, importance and therapeutic potential of these cells in EOC.

NK-T Lymphocytes
NK-T cells possess dual-functional capability: as T-cell subsets with a T-cell receptor (TCR)-mediated specific cytotoxicity and as NK cells with acquired killer functions [152,153]. NK-T cells have been found in increased frequencies in EOC tumor ascites compared to blood, but they were decreased at higher tumor grade and in cases of platinum resistance [154]. Moreover, the presence of NK-T cells was inversely correlated with VEGF ascites levels [155]. Since these cells display the most potent cytotoxicity profile, they might be promising agents for adoptive cell immunotherapy [156]. Further studies are needed to better understand the potential antitumoral capacity of these cells and their role in the different EOC TMEs.

NK Cells
Many studies have reported the presence of innate immune cells such as NKs, macrophages and DCs playing important roles in EOC tumorigenesis [103,124,154]. NK cells are crucial effectors in cancer immunosurveillance, recognizing and spontaneously killing virus-infected cells, cancer, and foreign cells hazardous to the host [157]. NK cells mediate antitumoral responses by secreting pro-inflammatory cytokines and chemokines such as IFNγ, TNF, IL-6, GM-CSF and CCL5, which influence antitumor activity and promote innate and adaptive responses in the TME [157][158][159] ( Figure 1). Tsiatas et al. analyzed 45 fresh specimens from different EOC and found an increased amount of CD56+ NK and NK-T cells along with activated CD4+ and CD8+ CD25+ T cells in serous and endometrioid carcinomas compared with mucinous and clear cell carcinomas [124]. Despite the high concentration of NKs found in ascites compared to peripheral blood, they are functionally impaired [121,160,161]. The influence of infiltrating NK cells on patient outcome is also debated. Analysis of ovarian carcinoma effusions showed that the presence of NK cells at an advanced stage (IV) predicted worse overall survival [151]. However, a positive antitumoral role for NK cells along with effector CD8+ T cells has been reported [162], and NK cell activity of peripheral blood lymphocytes was related to a significant progression-free survival of EOC patients [163]. Importantly, NK cells are activated or not, according to the balance between inhibitory and activating signals through different NK receptors [157]. Like many other cancers, EOC tumors express NK cell receptor ligand ULBP2, which is an indicator of poor prognosis and could promote T-cell dysfunction in the TME [164] ( Figure 2). Since NK cells are important players in antitumoral immunity, more studies aiding to characterize their function, phenotypes, incidence and role in the EOC TME are needed to provide new rational for immunotherapies.

Tumor-Associated Macrophages
Both TAMs and MDSCs constitute up to 20% of the EOC TME and are known to maintain and promote an immunosuppressive TME [103] (Figure 2). TAMs are considered the most abundant infiltrating immune cells in EOC tissue and ascites [165,166]. They possess an immunosuppressive M2 phenotype characterized by the expression of CD163, CD204, CD206, and IL-10 [165], and their presence correlates with tumor progression [140,167]. M2 TAMs secrete colony-stimulating factor 1 (CSF-1) that has been found in high levels in malignant EOC [167], and contributes to tumor growth, invasion, and metastasis. Moreover, EOC cells are able to induce an M2 TAM phenotype [168]. TAMs produce the chemokine CCL22 enabling the trafficking of Tregs to the ovarian tumors [130]. EOC cells as well as TAMs are known to express the coinhibitory molecule B7-H4 [169], a member of the B7 family that has a profound inhibitory effect on the growth, cytokine secretion, and development of T-cell cytotoxicity [169]. B7-H4+ TAMs are able to suppress antitumoral responses in EOC [136]. A study of 103 EOC patients showed that enhanced B7-H4 expression in macrophages correlated with Treg cell numbers in the tumor [136]. Tregs and B7-H4+ TAMs were associated with poor patient outcome. Tregs in the TME can induce B7-H4+ TAMs to produce IL-10 and IL-6 [136], further supporting an immunosuppressive milieu. Higher tumor grade correlated with higher frequencies of CD163+ TAMs [140] and worse progression-free survival [170,171]. Importantly, two studies evaluating M1-(HLA-DR, iNOS) and M2-polarization (CD163, VEGF) markers showed that higher M1/M2 TAMs ratio in tumors was associated with a favorable overall survival [172,173], and high serum levels of CD163 predicts poor EOC patient prognosis [174]. In addition, monocyte-derived macrophages in EOC displayed an altered morphology and defective antitumoral functions including defective antibody-dependent cell-mediated cytotoxicity and phagocytosis [175]. Thus, EOC cells and the TME provoke and maintain a strong immunosuppressive M2 phenotype supportive of tumor progression. Immunotherapeutic approaches aiming to switch TAM phenotypes [176] could help the evolution of antitumoral responses and improve patient outcome.

Myeloid-Derived Suppressor Cells
MDSCs are composed of a heterogeneous population of immature myeloid cells that arise in pathologic conditions such as cancer, inflammation and infection, and possess a potent capacity to dampen T-cell responses [177]. MDSCs are considered key inducers of tumor immune evasion and impaired immunity by upregulating arginase-1, nitric oxide, and reactive oxygen species, and by generating reactive nitrogen species [178] (Figure 2). MDSCs also deplete cysteine, induce Tregs, inhibit T-cell activation and proliferation, attenuate the cytolytic ability of NK cells, and trigger a M2 phenotype [103]. Obermajer et al. showed that the frequencies of CD11b+ CD14+ CD33+ CXCR4+ MDSCs in EOC ascites correlated with CXCL12 and prostaglandin E(2) levels [179]. MDSCs derived from EOC patients also increased gene expression of cancer stem cells, sphere formation and metastasis of EOC [180]. Wu et al. characterized typical monocytic CD14+ HLA-DR -/lo MDSCs in peripheral blood and ascites derived from EOC patients and found that MDSCs are enriched in both compartments [181]. Moreover, the density of MDSCs correlated with poor patient prognosis and elevated levels of IL-6 and IL-10 [181,182]. VEGF expression in EOC induced MDSCs recruitment, inhibiting local immunity [182]. A recent study with mouse EOC cells found that Snai1, a major transcription factor that induces epithelial-mesenchymal transition (EMT), mediates EOC progression by upregulating CXCR2 ligands, enabling the recruitment of MDSCs [183]. EOC cells also attracts myeloid cells by producing adenosine [184]. Hence, strategies targeting MDSCs could release the brakes against antitumoral responses. Metformin, a drug used to treat type 2 diabetes, may trigger EOC clinical benefit by improving antitumoral T-cell responses that are impeded by MDSCs in the TME, since this drug can block MDSC suppressor functions by decreasing CD39 and CD73 expression [185].

Exosomes
Highly proliferating cells such as cancer cells produce large amounts of exosomes which are small (40-100 nm) extracellular vesicles [186]. EOC tumor-derived exosomes carry cell membrane proteins and cargo proteins that could be used for diagnostics (EP-CAM) and immunotherapeutic targeting such as neoantigens and TAAs (Her2-neu, CA-125) [186], proteins (TGF-β1) [187], and miRNAs (miR-21) [188] that are involved in disease progression, metastasis, and chemoresistance [186], as well as immunomodulatory proteins (FAS-L) [189]. Exosomes can be taken up by other cancer cells, CAFs, and immune cells, therefore playing an important role in intercellular communication. Thus far, 2035 exosome cargo molecules have been identified from EOC cells in ExoCarta, a database for exosome cargo [190,191]. Exosomes derived from human patient ascites promotes tumor progression in vivo [189,192], and are proposed to have direct and indirect roles in modulating the immune TME, as exosomes could also be taken up by NKs and B cells [192] (Figure 2). In other disease models, such as melanoma and colorectal cancer, exosomes mediate immunosuppression and immune tolerance by suppressing the activation of T and NK cells, monocytes, modulating T-cell inhibitory molecules expression, and inducing CD8+ T-cell apoptosis [193,194]. FAS-L and TRAIL expression on EOC-derived exosomes inhibit activation of peripheral blood mononuclear cells by DCs through induction of apoptosis [189]. EOC-derived exosomes express ligands (MICA/B and ULBP1-3) for the NK receptor NKG2D, acting as a decoy and interfering with NK-mediated targeting of tumor cells [195]. Greater understanding of the complex network of the intercellular communication between EOC cells, CAFs, and immune cells is needed for the rational design of immunotherapeutic interventions, or leveraged for nanomedicine applications such as TAA loaded-DC-derived exosomes [196] and drug delivery systems [186].

Cancer-Associated Fibroblasts
CAFs are activated fibroblasts that express α-smooth muscle actin and fibroblast activation protein. They make up 7-85% [197] of the tumor and are the primary stromal cell type in the TME. Cross-talk between epithelial and stromal compartments creates a positive feedback loop, a supportive hyper-activated storm of cytokines, chemokines, angiogenetic factors, and EMT-promoting factors, to promote tumor progression and chemoresistance ( Figure 2). CAFs from ovarian cancer patients secrete high levels of hepatocyte growth factor (HGF) that promotes cancer cell proliferation, chemoresistance, invasion, and migration though constitutive activation of cMet/PI3K/Akt pathways and glucose-regulated protein 78 (GRP78) [198,199]. CAFs produce pro-inflammatory cytokines COX-2 and CXCL1 [200], CCL5 [201], CXCL11 [202], and IL-6 [203], which can promote proliferation and EMT. In addition to their direct actions on cancer cells, CAFs also produce exosomes with high levels of TGF-β1 that subsequently activates normal fibroblasts [187]. Interestingly, Givel et al. identified four CAFs subsets in HGSC, finding an accumulation of one subset, CAF-S1, in the mesenchymal molecular subtype of HGSC. CAF-S1 is associated with an immunosuppressive TME, due to its high levels of expression of CXCL12β, which recruits Tregs to the tumor. The CAF-S1 cells also express CD73, B7-H3 and IL-6, which subsequently promote survival and proliferation of Tregs [204]. Thus, CAFs can make major contributions to the creation of an immunosuppressive TME.
On the other hand, EOC cells can stimulate the activation of CAFs by producing high levels of interleukin-1β (IL-1β) [205] and TGF-β [206], which subsequently induces secretion of IL-8, IL-6, IL-1β, VEGF, and growth regulated oncogene-alpha (GRO-α) by CAFs to promote tumor progression [205]. EOC cells release exosomes not only to activate tumor cells, but also to reprogram normal fibroblasts into CAFs [207]. Furthermore, CAFs act on endothelial cells via the secretion of VEGF-C [208] or by upregulating genes such as lipoma-preferred partner, to promote angiogenesis, which leads to tumor progression and chemoresistance [209]. Cross-talk between CAFs and cancer cells, as well as endothelial cells and immune cells, suggests that targeting signaling mechanisms in this relationship may combat chemoresistance and immune modulation better than singly targeting the epithelial compartment.

Adipocytes and the Omentum
The unique TME of the omentum, a large visceral fat pad that covers the bowel and abdomen cavities [210,211], suggests a two-step model of omental metastasis and tumorigenesis where ovarian cancer cells preferentially and rapidly home to "milky spots" [212] in the omentum, prior to spreading throughout non-"milky spot" areas of the omentum and peritoneal cavity [213][214][215][216][217]. "Milky spots" are highly vascularized regions with aggregates of immune cells, capable of innate and adaptive immune functions, and antigen presentation similar to lymph node structures [212]. The involvement of the omentum and adipose tissue suggests the need to develop intraperitoneal immunotherapy similar to the advances seen with intraperitoneal chemotherapy.
Adipocytes in the omentum produce cytokines and chemokines, including highly secreted IL-6, IL-8, MCP-1, tissue inhibitor of metalloproteinases-1 (TIMP-1) and adiponectin, to promote cancer growth and omental metastases. Adipocytes can alter their lipid metabolism via Fatty acid-binding protein 4 (FAB4) to undergo lipolysis providing fatty acids (FA) to cancer cells as a fuel source for rapid tumor growth [216]. Cancer cells themselves can also alter lipid metabolism, often by upregulating FA receptor CD36 [218] and FAB4 in omental metastases at the tumor/adipocyte interface to promote FA and cholesterol uptake from adipocytes [216] to fuel tumor progression.
Many studies have suggested an association between obesity and the incidence of ovarian cancer as well as an association with poor prognosis [219]. Indeed, in a murine model of ovarian cancer, metastasis and tumor growth is supported in obese mice through altered regulation of FA pathway and increased immunosuppression, demonstrated by a decreased ratio of M1/M2 macrophages [220] ( Figure 2). Improved understanding of how adipocytes and the omentum support ovarian cancer growth and promote peritoneal metastases will reveal therapeutic targets for both conventional therapy and immunotherapy. It will be important to consider how age and obesity [221][222][223] may dictate differences in response to immunotherapy and how current models with young, lean mice may fail to accurately model responses to immunotherapy.

Conclusions
In summary, in order to develop better immunotherapies for EOC we need to identify and consider all key elements found in the TME of not only primary tumors but also in ascites and metastases with a focus on how these features affect and are affected by different cancer therapies. It is crucial to take into account the quality of the TME (immune-activating vs. immune-suppressing mechanisms), tumor immunogenicity, tumor burden mutations, tumor stage, patient overall condition, and age, as well as treatment effects on the TME (chemotherapy, neoadjuvant chemotherapy, surgery debulking). Each of these factors may influence the outcome of EOC and the responses to cancer immunotherapies. Moreover, to avoid tumor recurrence, EOC characteristics such as TAA presentation, expression of coinhibitory molecules, production of immunosuppressive cytokines and chemokines should all be considered to find therapeutic combinations that could synergize and achieve maximal benefits to eliminate EOC. Other articles in this special issue will address some of these topics, including the exploration of promising immunotherapies for HGSC that are currently under investigation [224].
Author Contributions: All authors contributed to the research, writing, and editing of this review article.
Funding: The authors' research relevant to this article is funded by grants to BCV from BioCanRx and the Canadian Institutes of Health Research, as well as a fellowship from the Fonds de recherche du Québec-Santé (GMR), and a Vanier Canada Graduate Scholarship (CM).