Aggressive Signals Research Articles

Call for Your Life: Acoustic Structure and Age‐Sex Differences in Distress Calls of Red‐Necked Nightjars

ABSTRACTPredatory interactions result in strong selection pressures acting on multiple aspects of animal behaviour. Anti‐predator strategies are therefore common in most animals, typically signalling at various stages of a predation event. Many species of caprimulgids perform conspicuous anti‐predator displays, including stereotyped behaviours and vocal signals. Here, we described distress calls of red‐necked nightjars (Caprimulgus ruficollis), produced when birds are trapped and unable to escape during a predatory interaction. Distress calls are harsh, low‐frequency guttural vocalisations with irregular amplitude modulations. The age and sex of birds partially explained the acoustic variation observed, whereas size‐related morphological features were poor predictors of the acoustic structure. Age‐sex differences in distress calls may respond to physiological constraints associated with sexual dimorphism and/or developmental variation. Alternatively, directional selection associated with differential predation risk on each age‐sex class may have resulted in the observed differences in distress calls. The extremely deep voice and the harsh quality of distress calls fit the structure of aggressive signals and may resemble those produced by a larger animal. We propose that these calls serve as a last resort strategy to reduce post‐encounter risk of predation, either as a startling effect to facilitate escape or to attract other predators that could intimidate the captor.

Changes to the acoustic properties of Gromphadorhina portentosa defensive sounds when exposed to the molting hormone, 20‐hydroxyecdysone

AbstractSteroid hormones play a pivotal role in shaping arthropod phenotypes, with 20‐hydroxyecdysone (20E) serving as a key regulator of molting, a vulnerable period in an insect's lifecycle. Despite its critical role in arthropod growth and development, the influence of 20E on arthropod behavior, particularly defensive strategies, remains poorly understood. We investigated the impact of 20E on the bioacoustic characteristics of hisses in the Madagascar hissing cockroach (Gromphadorhina portentosa), a social species with multiple complex acoustic signals. With increased 20E, we predicted that hiss production would be more likely and more defensive (i.e., longer hisses with greater intensity (dB) and reduced frequency (Hz)). We injected male G. portentosa with either a low‐ (35 μg) or high‐dose (70 μg) of 20E or a control (0 μg 20E), and we measured the presence/absence of hissing responses and their bioacoustic characteristics following a standardized tactile stimulus. Contrary to our prediction, there was no difference in the likelihood of hissing or hiss duration with 20E administration. However, administering 20E resulted in reduced hiss intensity and increased hiss frequency (as measured by peak and center frequencies), suggesting potential shifts from defensive to aggressive signaling. Our study contributes to the limited knowledge of the behavioral effects of 20E, suggesting that some arthropods may experience increased aggression or energetic limitations to defense during molting. Behavioral changes elicited by hormones have important implications for both fundamental ecology and applied pest management.

The evolution of honest and dishonest signals of fighting ability.

Competition over resources is often decided via aggressive interactions, which may or may not escalate to all-out fights. Weapons and body size play important roles in such interactions, as they often provide reliable cues of an individual's fighting ability. In contrast, traits like nonfunctional display "weapons" may dishonestly exaggerate fighting ability in order to intimidate opponents into retreating. Signals used in the context of aggressive interactions potentially evolve via very different mechanisms than courtship signals, but have received far less theoretical attention. Here, we contrast the evolution of honest and dishonest signals of fighting ability using a game-theoretic model. Contests are assumed to consist of three discrete stages: display from a distance, low-intensity physical contact, and fighting. At each stage, contestants evaluate the fighting ability of their opponents in comparison to their own based on body size and an aggressive signal. After making this evaluation, contestants decide whether to escalate the interaction or cede to their opponent. Our model predicts that both honest and dishonest aggressive signals can exaggerate far beyond their ecological optima, but that exaggeration is more pronounced for honest signals. Equilibrium levels of aggressiveness-as measured by individuals' propensity to escalate aggressive interactions to the next stage-are independent of the honesty of signals. We additionally develop a novel approach, based on causal inference theory, to understand how changes in underlying parameters shape the coevolution of multiple traits. We use this approach to study how aggression coevolves with body and signal size in response to changes in the cost of losing a fight.

Cross-context behavioural correlations and signals of aggression in females of a livebearing fish

Abstract Behaviours can be adaptively correlated with each other and with other aspects of phenotype. We investigated behaviour across foraging, mating, and risk contexts in females of the poeciliid fish, Girardinus metallicus. We quantified relationships between these behaviours and aggression signals, body size, and reproductive output. Behaviours describing aggression and boldness, some of which were repeatable, were correlated in females. Aggression was signalled by the darkening of a black spot on the extended dorsal fin or by fin flaring. Spot darkening occurred during intra- and intersexual interactions and was positively correlated with interfemale aggression and negatively correlated with the time females spent following males, suggesting that it is an honest indicator of motivational state. In contrast, fin flaring was directed only at females and occurred more frequently in the foraging context. Larger females had fewer offspring and received fewer courtship displays, possibly because they were more aggressive to males. In contrast to studies of males, we found a negative relationship between rank order of boldness and aggression, consistent with either a trade-off or with selection favouring the negative relationship. Our results highlight the importance of studying females in model systems to uncover novel patterns and potentially meaningful departures from what is typically seen in males.

Buzzes are used as signals of aggressive intent in Darwin’s finches

Abstract Signals of aggression may potentially reduce the fitness costs of conflict during agonistic interactions if they are honest. Here we examined whether the ‘buzz’ vocalization in two species of Darwin’s finches, the small tree finch, Camarhynchus parvulus, and the critically endangered medium tree finch, C. pauper, found in Floreana Island, Galápagos Archipelago, is a signal of aggression. Specifically, we assessed three criteria for aggressive signalling (context, predictive, and response criteria) in an observational study and a playback experiment. In the observational study, buzzes by the resident male were more common when an intruder was present on the territory in medium tree finches but not small tree finches (context criterion). In the playback experiment, buzzes increased during and after a simulated intrusion for both species (context criterion). Buzzes before the playback period predicted aggressive responses by males (predictive criterion) but buzzes during playback did not. Finally, both species responded more strongly to playbacks of conspecific buzzes compared to conspecific songs and heterospecific buzzes (response criterion). Together the results support the aggressive signal hypothesis for buzz vocalizations, although future studies are needed to understand the evolution and development of this interesting signal.

Cultural facial expressions dynamically convey emotion category and intensity information

Communicating emotional intensity plays a vital ecological role because it provides valuable information about the nature and likelihood of the sender's behavior.1,2,3 For example, attack often follows signals of intense aggression if receivers fail to retreat.4,5 Humans regularly use facial expressions to communicate such information.6,7,8,9,10,11 Yet how this complex signaling task is achieved remains unknown. We addressed this question using a perception-based, data-driven method to mathematically model the specific facial movements that receivers use to classify the six basic emotions-"happy," "surprise," "fear," "disgust," "anger," and "sad"-and judge their intensity in two distinct cultures (East Asian, Western European; total n= 120). In both cultures, receivers expected facial expressions to dynamically represent emotion category and intensity information over time, using a multi-component compositional signaling structure. Specifically, emotion intensifiers peaked earlier or later than emotion classifiers and represented intensity using amplitude variations. Emotion intensifiers are also more similar across emotions than classifiers are, suggesting a latent broad-plus-specific signaling structure. Cross-cultural analysis further revealed similarities and differences in expectations that could impact cross-cultural communication. Specifically, East Asian and Western European receivers have similar expectations about which facial movements represent high intensity for threat-related emotions, such as "anger," "disgust," and "fear," but differ on those that represent low threat emotions, such as happiness and sadness. Together, our results provide new insights into the intricate processes by which facial expressions can achieve complex dynamic signaling tasks by revealing the rich information embedded in facial expressions.

Experimental manipulation of chest spotting alters territorial aggression in urban and rural song sparrows

In many taxa, melanin-based coloration is a signal of dominance or fighting ability and is associated with concentrations of hormones that may mediate aggressive behavior. Previous studies found that experimental manipulation of melanin-based signals can result in manipulated individuals receiving more social challenges in some but not all species. These differences could arise from mismatches between the signal, behavior, and hormone concentrations. In the present study, we experimentally manipulated the chest spotting of urban and rural male song sparrows (Melospiza melodia) following an assessment of their territorial aggression and initial concentrations of corticosterone and testosterone and then assessed their behavior and hormone concentrations 2 weeks later. We found that males generally displayed less territorial aggression in the second trial, consistent with our previous findings. Males in the enlarged treatment decreased aggression to a greater degree than those in the reduced treatment. The effect of the plumage manipulation was similar across the rural and urban habitats. Despite the changes in behavior we detected, we found no effects of the manipulation on concentrations of testosterone or corticosterone. Our results show that melanin-based spotting in male song sparrows is a signal of territorial aggression but the physiological mechanisms that mediate the relationships between chest spotting and behavior remain to be identified.Significance statementMany bird species use their plumage to signal their dominance status, fighting ability, or motivation during interactions with other individuals to resolve conflicts without a fight. Here, we asked whether chest spotting is a signal in territorial interactions among male song sparrows. We experimentally increased or reduced the extent of spotting in males and measured the change in their aggression. We found that reduced-spotting males showed a more moderate seasonal decrease of aggression compared to males with enlarged spotting reduced aggression, possibly because the former experienced more intrusions later on in the breeding season while the latter experienced fewer intrusions. These results are consistent with chest spotting size in song sparrows functioning as a signal of territory holding potential of the bearer.

Comparative Study of Aggressive Signaling in Three Closely-Related Warbler Species

Comparative Study of Aggressive Signaling in Three Closely-Related Warbler Species

Color as an Interspecific Badge of Status: A Comparative Test.

AbstractAnimals as diverse as cephalopods, insects, fish, and mammals signal social dominance to conspecifics to avoid costly fights. Even though between-species fights may be equally costly, the extent to which dominance signals are used between species is unknown. Here, we test the hypothesis that differences in color are associated with dominance between closely related species that aggressively interact over resources, examining between-species variation in colors that are used in within-species badges of status (black, white, and carotenoid coloration) in a comparative analysis of diverse species of birds. We found that dominant species have more black, on average, than subordinate species, particularly in regions important for aggressive signaling (face, throat, and bill). Furthermore, dominant species were more likely to have more black in comparisons in which the dominant species was similar in size or smaller than the subordinate, suggesting that black may be a more important signal when other signals of dominance (size) are missing. Carotenoid colors (i.e., red, pink, orange, and yellow) were not generally associated with dominance but may signal dominance in some taxonomic groups. White may have opposing functions: white was associated with dominance in species in which black was also associated with dominance but was associated with subordinance in species in which carotenoid-based dominance signals may be used. Overall, these results provide new evidence that colors may function broadly as signals of dominance among competing species. Such signals could help to mediate aggressive interactions among species, thereby reducing some costs of co-occurrence and facilitating coexistence in nature.

Sexual Orientation Affects Neural Responses to Subtle Social Aggression Signals

The current series of studies are the first to examine brain responses to social aggression signals as a function of male and female sexual orientation. For the first set of studies (1a, 1b), axillary sweat had been collected from 17 heterosexual men and 17 heterosexual women aggressively responding to frustrating opponents (aggression condition) and while playing a construction game (control condition). Sweat samples were pooled according to sex and condition, and presented via a constant flow olfactometer to 17 gay and 23 heterosexual men (Study 1a), and 19 lesbian and 25 heterosexual women (Study 1b). Ongoing EEG was recorded from 61 scalp locations, chemosensory event-related potentials (CSERPs; P2, P3-1, P3-2) were analyzed, and neuronal sources calculated (low resolution electromagnetic tomography). Within the second set of studies (2a, 2b), pictures of males’ and females’ weak angry and neutral facial expressions were presented to 21 gay and 23 heterosexual men (Study 2a), and 19 lesbian and 26 heterosexual women (Study 2b), and ERPs (N170, P3) were analyzed. Gay men showed larger P3-1 amplitudes than heterosexual men upon presentation of male aggression sweat, accompanied by activation of the right inferior frontal gyrus (IFG, BA 10). Gay men also displayed longer N170 latencies in response to men’s compared to women’s angry facial expressions, while heterosexual men did not. In women, sexual orientation did not affect the processing of aggression sweat or anger expressions. Gay men showed preferential processing of chemosensory aggression signals (P3-1 amplitudes), indicating fine-tuned socioemotional sensitivity, related to activation of brain areas involved in emotion regulation (IFG). They further process the relative relevance of visual aggression signals (N170 latency). These results were in line with theories proposing a common evolutionary pathway for same-sex attraction and traits easing social integration.

Angry birds calling: an advanced system of signalling aggression to moderate conflict in the common chiffchaff

Many animals produce vocal signals during agonistic interactions that convey their ability and motivation to escalate the conflict. Often, such disputes follow ritualized, sequential phases with increasing aggression, favouring the assessment of individuals' chances of winning. This allows for individuals to withdraw before getting involved in a serious fight. Birds are well known for the variety of information encoded in their vocalizations, sometimes including specific features that signal aggression. A particular case may be so-called ‘tret calls’ produced by chiffchaffs, Phylloscopus collybita, a song affix for which the communicative role is not well understood. Here, we describe the acoustic structure and use of tret calls during simulated conflicts using playback. Our results show that tret calls are a low-amplitude vocalization and that birds increased tret calling after playback stimulation, suggesting a role as an aggressive signal similar to soft song in other species. However, we did not find that the tret call rate predicted subsequent aggressive behaviour. Our analysis of song variation in response to playback suggested that birds respond to a simulated vocal competitor in two phases: a highly aggressive phase where syllable rate goes up, which is predictive of physical attack, while simultaneously tret call rate goes down, and a low-aggression phase, in which tret call rate goes up, while syllable rate returns to baseline levels. These stereotypic fluctuations in two acoustic features may be part of an advanced communication system to moderate aggression. Finally, our results show a seasonal increase in tret calling after birds arrive at their breeding grounds, which is in line with a role in territorial aggression. We also found a nonsignificant trend for more tret calls with increasing ambient noise levels, which may be due to increased stress and aggression under noisy conditions or reflect a masking avoidance strategy.

Male song of the Aquatic Warbler, a promiscuous bird without paternal care, is more complex than previously thought

The Aquatic Warbler Acrocephalus paludicola is one of the rarest European passerines and is characterised by promiscuity, lack of pair-bonds and female-only parental care. This makes the species an important model for studying the function of avian courtship song. The song of the Aquatic Warbler consists of whistle and rattle phrases produced as discontinuous A-, B- and C-songs, which are built by a single rattle, a rattle and a whistle, and more than two phrases of both kinds, respectively. The A- and B-songs are thought to be aggressive signals in male-male interactions, while C-songs are thought to be important for female choice. Here, we analysed recordings of 40 individually marked males, and determined the phrase repertoire. The enumerated repertoire (males recorded for ≥ 10 min) ranged from 16 to 158 (mean 99), however, it did not capture the complete phrase repertoires. We then used models from species diversity ecology to estimate the actual phrase repertoire size, which ranged between 18 and 300 phrases (mean 155). The estimated repertoire was predicted by the number of C-songs. The rattle repertoire was larger than the whistle repertoire, and both positively correlated with the number of C-songs. Our study indicates that male Aquatic Warblers have highly complex phrase repertoires that vary widely in size. Their courtship song is flexible and efficient, enabling relative song complexity to be demonstrated in a short sample, thus facilitating both female attraction through the quick presentation of large phrase repertoires and rival deterrence through the production of many short and simple A- and B-songs.

Genetic correlation between aggressive signals and fighting.

Theoretical analyses indicate that aggressive signals should positively correlate with the signallers' willingness and abilities to fight. Few experimental studies, however, have tested this prediction. In two experiments employing distinct, ecologically realistic protocols, we quantified the association between aggressive signals and fighting in fruit fly genotypes and found high positive genetic correlations between threat and fighting (rG = 0.80 and 0.74). Our results add to the growing body of experimental work indicating that aggressive signals have relatively high informational value.

Soft calls do not modulate aggressiveness in male common cuckoos toward conspecific territorial intruders: A sequential playback study

In many bird species loud broadcast calls serve as aggressive signals with a large effective radius, whereas soft calls may indicate aggression at a closer distance, and are often directed at a nearby conspecific individual. Male common cuckoos (Cuculus canorus) are famous for their simple cu-coo calls, which are long-range broadcast calls, uttered in long sequences during the breeding season. Cuckoos also produce soft calls, including a single gowk (wah), a series of gowks (gowk series), or a harsher single variant, the guo call. To our knowledge, no previous study has attempted to understand the function of cuckoos’ soft calls. We conducted a sequential playback experiment by (i) first attracting male cuckoos with “cu-coo” calls, and then (ii) playing heterospecific control calls or one of several types of cuckoo soft calls. While the “cu-coo” call attracted focal males, neither the soft conspecific calls nor the heterospecific control calls elicited an additional response. Finally, the gowk series calls did not alter cuckoos’ approach to the speaker when played together with “cu-coo” calls. As cuckoos’ soft calls did not increase or decrease aggression, they instead may advertise the cuckoo’s presence and position for a nearby conspecific.

Structural-functional characteristics of two song types in Phylloscopus humei (Phylloscopidae)

Several songbird species use two singing modes, which are functionally different. The first mode is preferentially used at the beginning of the breeding cycle, and serves to attract females, while the second mode is used later in the season as well as during territorial countersigning. The two singing modes are well known in many Parulidae species from North America. The repeat mode (type I songs) comprises repetitions of a single song type. The serial mode (type II songs) consists of several song types sung in a versatile sequence. In Eurasia, a similar acoustic behaviour is known in several Phylloscopus species. However, these data are still scarce. Additionally, it is not yet fully understood whether the song structure per se or the song bout organisation (e.g. song-type diversity) plays a primary role in communication of the aforementioned songbirds. In this respect, it could be useful to analyse acoustic behaviour of Phylloscopus humei because its males have only two song types, namely song type I and song type II. These song types differ greatly in structure. In this study, we used playback experiments to ask whether these song types differ in their function. The study has been conducted in 2019–2021 in the federal State Nature Sanctuary «Posarym» (Republic of Khakassia, Russia) at the beginning of the breeding cycle of Phylloscopus humei. While singing spontaneously, males predominantly use song type I. The use of song type II is increased immediately after playback presentation. In contrast, males did not increase the rate of song type II during the playback. Instead, they produced two call types at that time, which apparently serves as an aggressive signal. Our study has shown that the use of two song types is different. Song type I is predominately used for advertising the territory and attracting a female. In contrast, song type II is more often used in countersigning between neighbouring males, although not at a time of direct aggression, i.e. during the playback. Noteworthy, we also found that males can share song type II but not song type I from their repertoires. In turn, the song sharing is thought to play a role in male-male interaction.

Subordinate submissive responses are predicted by dominant behaviour in a cooperatively breeding fish

Abstract In complex social groups, animals rely on communication to facilitate priority access to resources and minimise the costs of conflict. Animals typically have more aggression signals than submission signals. However, some social species do show multiple submission signals, and the context in which these different signals are used is often not well understood. In the current study, we assessed agonistic interactions within groups of the cooperatively breeding daffodil cichlid fish (Neolamprologus pulcher) to investigate the relationship between the aggressive behaviours of the dominant breeding pair, and the submissive responses of the highest ranked subordinate within the group. Daffodil cichlids may respond to aggression by fleeing or by the production of either a tail quiver display or a head up display. Among the two submission signals, the tail quiver display was used more frequently in response to a threat display, while head up displays were produced approximately equally in response to both threat displays and overt aggression. An exaggerated version of the head up display was given more often in response to overt aggression, suggesting a graded submissive response both within and between the two submission signals. Within fish, the frequency of head up displays, but not tail quiver displays, correlated positively with the frequency of threat displays received. The current study helps us to better understand the use of submission signals in a highly social vertebrate and sheds light on submission as an understudied aspect of communication.

Soft song provokes stronger aggressive responses than broadcast song in the Plain Laughingthrush (Pterorhinus davidi)

Many bird species produce low‐amplitude acoustic signals, known as soft songs, which are widely believed to function primarily in aggressive interactions. However, there are surprisingly few experimental studies that have examined whether soft songs are reliable signalling strategies for escalating aggressive interactions. Here, we investigate the functions of soft song in a colour‐marked population of the Plain Laughingthrush Pterorhinus davidi, using playback experiments combined with specimen‐based simulated territory intrusions. We found that playback of male soft song provoked quicker response and stronger aggressive intent by territorial pairs compared with playback of male broadcast song. Responses by territorial pairs to male soft song playback were significantly stronger compared with responses to broadcast song playback, with a greater number of flights over the speaker, more time spent within 1 m of the speaker and closer approaches to the speaker. Furthermore, closer approach with the production of soft song by focal males was the best predictor of aggressive intent in combined playback and specimen intrusion experiments. Our results suggest that male soft song is used as an aggressive signal in the Plain Laughingthrush, and represents the first evidence of such for a monogamous temperate duetting species.

What About Females? Urban Female Song Sparrows Elevate Aggressive Signaling Compared to Rural.

The costs and benefits of breeding behaviors are influenced by environmental conditions, and habitat variation can shift the degree to which behaviors are expressed. Novel urban habitats have been shown to differ significantly in disturbances such as noise, light at night, and human presence, as well as resource availability, compared to rural habitats. Perhaps because of these environmental differences, urban males of several species are consistently more aggressive than rural males, raising the hypothesis that greater territorial aggression is beneficial in urban habitats. Though often ignored, female songbirds of many species also perform aggressive territorial behaviors towards conspecifics during the breeding season. For socially monogamous songbirds, this aggression functions to ensure partner fidelity and secure resources for reproduction. Studies of the effects of urbanization on songbird behavior have yet to determine if urban females also express greater territorial aggression. Importantly, energetically demanding behaviors such as territoriality and parental care should constrain one another, leading to behavioral trade-offs during the breeding season. Though territorial aggression and parental care are inversely related in males of several species of songbird, this relationship is understudied in female songbirds, particularly those facing environmental change such as urbanization. In this study, we compared aggressive signaling and a measure of parental care (maternal nest visitation rates) between female song sparrows (Melospiza melodia), living in urban and rural habitats. We hypothesized that female aggressive signaling would be higher in urban environments compared to rural, and negatively correlated with maternal visitation rates. We found that urban females, like males, expressed increased aggressive signaling compared to rural. However, female aggressive signaling was not related to our measure of maternal care, suggesting females aren't facing a trade-off between these two behaviors. Collectively, our results are consistent with the hypothesis that urban habitats promote territorial aggression in female song sparrows. As urbanization continues to spread, understanding the behavioral changes animals employ in urban environments requires studying individuals of different sexes and age classes, and will help us understand how some species are able to cope with human induced rapid environmental change.

Abstract 1042: Fc3TSR improves intratumoral treatment delivery by normalizing tumor vasculature and reducing interstitial fluid pressure in an orthotopic, syngeneic mouse model of epithelial ovarian cancer

Abstract A hallmark of solid tumors is the need for vascularization to supply oxygen and nutrients. Aggressive pro-angiogenic signals induced by tumors lead to malformation of vessels characterized by reduced pericyte coverage and low perfusion. As a result of this vascular dysfunction, tumors have elevated hypoxia and high interstitial fluid pressure (IFP), yielding an aggressive phenotype, and reducing efficacy and trafficking of intravenous therapies to the tumor core. Anti-angiogenic therapies aim to reduce angiogenic stimuli and normalize tumor vessels. The three type-1-repeat (3TSR) region of thrombospondin-1 contains the majority of its anti-angiogenic properties in a small bioactive peptide. These functions are mediated through the membrane protein, CD36. We have previously shown that administration of native 3TSR leads to improved tumor perfusion, and enhancing delivery of chemotherapy drugs and oncolytic viruses. We have developed a novel compound, Fc3TSR, that has two 3TSR peptides linked with a Fc to increase in vitro and in vivo efficacy. In an orthotopic, mouse model of ovarian cancer, we examined the effect of Fc3TSR treatment on tumor IFP using a 1.2Fr pressure catheter guided into the tumor core of anesthetized mice. Compared to untreated controls, mice treated with Fc3TSR had significantly lower IFP, even after considering differences in heart rate among animals. Immunofluorescence on fixed sentinel lymph nodes revealed enhanced presence of immune cells, a positive predictor of lymphatic patency. To determine impact on chemotherapy delivery, mice were injected with 40mCi paclitaxel and tumor uptake of the isotope was measured. At 12, 24 and 48h post injection, Fc3TSR treated tumors had significantly increased uptake of paclitaxel compared to untreated or 3TSR treated mice. In vitro cell viability assays and western blot analysis of cleaved-caspase-3 revealed that Fc3TSR enhances direct apoptosis in human ovarian cell lines (OVCAR-2 and 36M2) compared to untreated cells or those treated with native 3TSR at equimolar concentrations. In a human subcutaneous xenograft model using 36M2 cells in SCID mice, Fc3TSR significantly reduced tumor volume as a single agent compared to PBS controls.Fc3TSR causes direct ovarian tumor cell apoptosis in vitro and significant anti-tumor effects in vivo. In vivo, Fc3TSR causes potent vascular normalization, decreases tumor hypoxia and IFP in the tumor core. As such, Fc3TSR has the potential to remodel multiple aspects of the tumor microenvironment which would otherwise obstruct treatment delivery and efficacy in solid tumors. The multi-modal aspects of Fc3TSR makes this therapeutic approach attractive for the treatment of advanced ovarian cancer and other malignancies that typically overcome single-agent therapy. Citation Format: Kathy Matuszewska, Madison Pereira, Leslie Ogilvie, Duncan Petrik, Allison Gartung, Dipak Panigrahy, Kin-Ming Lo, Jack Lawler, Jeremy Simpson, Jim Petrik. Fc3TSR improves intratumoral treatment delivery by normalizing tumor vasculature and reducing interstitial fluid pressure in an orthotopic, syngeneic mouse model of epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1042.

Abstract 1523: Beta-2 adrenergic receptor and mu opioid receptor interact to increase the aggressiveness in triple negative breast cancer cells through activation of GSK3 signaling

Abstract Breast cancer is the world’s leading cause of cancer mortality in women worldwide. It has been shown that body stress hormones, such as norepinephrine, and opioid peptides interact to control the growth and progression of this cancer. Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control cellular functions in various cell systems. However, the underlying cell signaling mechanism remains unclear. To determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and MDA-MB-468, CRISPR, chemical inhibition or activation and, PCR Array technology to study aggressive tumor phenotype and signaling cascades. We found that knock down of the beta-adrenergic (B2AR) or mu-opioidergic receptor (MOR) system via the CRISPR technique or pharmacological agents induces a less aggressive phenotype in triple negative breast cancer cells both in vitro and in vivo, with the effect being potentiated when both receptor systems were inhibited together. The mRNA expression study using a PCR Array targeting Cancer Stem Cell genes shows that B2AR or MOR activated the glycogen synthase kinase 3 (GSK3) signaling cascade. The GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as was done by the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation. These data identify a role of B2AR and MOR interaction in the control of breast cancer cell growth and establish a possible role of the GSK3 signaling system in mediation of these two receptors’ cross talk. (Supported by NIH grant R01 CA20863201). Citation Format: Bénédicte Rousseau, Sengottuvelan Murugan, Ajay Palagani, Dipak K. Sarkar. Beta-2 adrenergic receptor and mu opioid receptor interact to increase the aggressiveness in triple negative breast cancer cells through activation of GSK3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1523.