Reproductive coercion is a significant public health issue in Australia which has mainly been conceptualised as a form of violence at the interpersonal level. This limited scope ignores the role of the gendered drivers of violence and fails to encompass a socio-ecological lens which is necessary to consider the multiple interacting layers that create the context in which reproductive coercion occurs. The aim of the scoping review was to explore how the reproductive coercion is defined by international research. Specifically, how is reproductive coercion defined at the social-cultural-systems-structural levels, and are the definitions of reproductive coercion inclusive of the conditions and contexts in which reproductive coercion occurs?A scoping review was undertaken to explore existing definitions of reproductive coercion. Searches were conducted on Embase, Cochrane Library, Informit Health Collection, and the EBSCOHost platform. Google was also searched for relevant grey literature. Articles were included if they were: theoretical research, reviews, empirical primary research, grey literature or books; published between January 2018 and May 2022; written in English; and focused on females aged 18–50 years. Data from eligible articles were deductively extracted and inductively thematically analysed to identify themes describing how reproductive coercion is defined.A total of 24 articles were included in the scoping review. Most research defined reproductive coercion at the interpersonal level with only eight articles partially considering and four articles fully considering the socio-cultural-systems-structural level. Thematic analysis identified four main themes in reproductive coercion definitions: Individual external exertion of control over a woman’s reproductive autonomy; Systems and structures; Social and cultural determinants; and Freedom from external forces to achieve reproductive autonomy.We argue for and propose a more inclusive definition of reproductive coercion that considers the gendered nature of reproductive coercion, and is linked to power, oppression and inequality, which is and can be perpetrated and/or facilitated at the interpersonal, community, organisational, institutional, systems, and societal levels as well as by the state.The online version contains supplementary material available at 10.1186/s12889-023-16281-8. Reproductive coercion is a significant public health issue globally, driven by gender inequality and imbalances in power, with negative consequences for a wide range of sexual, reproductive, and mental health issues –2. It isGiven the historically hidden nature of reproductive coercion, limited Australian research exists to demonstrate its full extent. The data that does exist relates to women’s experiences of gender-based violence, which commonly includes reproductive coercion. Research exploring the prevalence of violence against women in Australia demonstrates that one in five women over the age of 15 will experience violence in their lifetime . FurtherThe categorisation of reproductive coercion as a form of violence , 18 has Reproductive coercion has historically been the responsibility of multiple, overlapping sectors. These include the domestic violence, sexual assault/harassment and sexual and reproductive health sectors which are informed by their own distinct approaches to prevention, policy reform and service delivery . A recenA socio-ecological approach can be utilised to move beyond the interpersonal level. This approach recognises the broader systems that individuals are embedded within and the changing nature of the social environment . Socio-eThe model highlights how violence against women is the outcome of interactions between various levels (particularly at the primary prevention level), therefore it uniquely moves beyond just looking at the interpersonal level (or tertiary prevention level). The model was developed to support public health practitioners and academics to approach violence against women from a primary prevention lens, which requires underlying drivers to be addressed across all socio-ecological levels. Like other forms of violence against women, we argue reproductive coercion is a complex public health issue that needs to be viewed and addressed from a socio-ecological lens, as opposed to only focusing on the interpersonal level.It is clear reproductive coercion within the Australian context is multi-layered, complex, gendered, driven by gender inequality and differentials in power, and rooted in historical experiences yet there has been no exploration of how definitions of reproductive coercion may be limiting the scope of work to prevent and address reproductive coercion. As such, the current scoping review explored existing definitions of reproductive coercion, drawing on international research due to the limited Australian research, and asked; how is reproductive coercion defined, how is reproductive coercion defined at the social-cultural-systems-structural levels, and are definitions of reproductive coercion inclusive of the conditions and contexts in which reproductive coercion occurs? This approach enabled an exploration of reproductive coercion in the broadest sense to enable alternative definitions and contexts to be considered in how we might better conceptualise reproductive coercion in Australia. The outcome of this review is a proposed definition of reproductive coercion that moves beyond the interpersonal and considers the role of gendered drivers at the societal, system, institutional, organisational and community levels is conceptualised, which may be applicable in other similar socio-cultural-politico contexts to Australia.A scoping review was undertaken following the process as described by Arksey and O’Malley , and advFive authors  developed the search strategy, search terms and eligibility criteria, which were refined after preliminary searches were completed (AD). The information sources searched included Embase, Cochrane Library, Informit Health Collection, and the EBSCOHost platform , PsycINFO (psychology), and SocINDEX (sociology)). Google was also searched for grey literature.Search terms covered two key concepts: 1) reproductive coercion; and (2) definition  and combined using the OR operator and repeated for concept 2 (definition). Concepts 1 and 2 were then combined using the AND operator to conduct the final search. For example, “reproductive coercion” OR “coercive reproduction” OR “reproductive pressure” OR “reproductive control” OR “reproductive sabotage” OR “reproductive autonomy” OR “reproductive agency” AND definition OR conceptualise OR framing OR theory OR defining OR theoretical OR lens OR positioning OR constructs OR elements OR attributes. Searches were set to identify key terms in the titles and abstracts.A search was also undertaken using Google to identify any grey literature. The Google search was restricted to .gov, .org, or .edu domains and was conducted using the same search terms Table . The firTo be eligible for inclusion articles had to be: theoretical research articles, reviews , empirical primary research, grey literature , books, commentaries or opinion pieces; published between January 2018 and May 2022; written in English; full-text available; focus on females of reproductive age (18 to 50 years); and include a definition of reproductive coercion. Dissertations and conference presentations were excluded. The search was limited to articles published between January 2018 and May 2022 to ensure contemporality of the evidence.While reproductive coercion is not limited to cis-gendered females of reproductive age, research focusing only on adolescents was excluded. However, research which included both adolescent and adult females was included. The reproductive age span selected (18 to 50 years of age) reflects when adult females are most likely to be directly impacted by and experience reproductive coercion. Reproductive coercion may also occur among transgender and gender diverse people. While non-cis-gendered female search terms were not included, the term female was not considered exclusively as meaning cis-gendered female and as such did not exclude adult female identifying populations. To this end, females were not considered exclusively as cis-gender, and reproductive age sometimes included females younger than 18 years alongside those aged 18 years and over.Results from the searches were uploaded into Covidence. All publications were screened for eligibility. Abstracts were reviewed against the eligibility criteria by any two of the researchers. In cases of a disagreement, a third researcher reviewed the abstract which was then discussed to reach a consensus. Publications which met the eligibility criteria based on abstract screening were then moved to full-text screening. As per abstract screening, the full-text of the publications were reviewed against the eligibility criteria by any two members of the research team, with a third reviewing the full-text when there was a conflict. The three researchers then discussed the publication to reach a consensus regarding its inclusion or exclusion. The main reason for conflict was whether key findings related to defining reproductive coercion.Data extraction was undertaken for each article including author(s), year of publication, publication type, aims/objectives, study design, sampling method(s) or eligibility criteria, data collection methods, study population (sample size and characteristics), key findings related to the research questions, conclusion/recommendations, and limitations. An abbreviated data extraction chart is provided in supplementary Table Data were thematically analysed to identify key ideas, concepts, and factors relevant to the research questions. These were then reviewed to identify any similarities and were merged to develop themes. While data extraction provided a way to draw out data, thematic analysis provided a more nuanced and contextual understanding of the ways in which reproductive coercion is defined and we believe, more informative insights than a profile of the included studies as a data chart , 28. ThrThe search strategy produced 239 articles once duplicates were removed. Of the 239 articles, title and abstract screening against eligibility criteria excluded 192 articles. At full-text screening, 47 articles were assessed against eligibility criteria and 23 were excluded. Of the 23 excluded articles, 22 had no key findings related to defining reproductive coercion, and one article was an excluded source type (conference abstract). A total of 24 articles were included in the scoping review. Figure All 24 articles included study populations that focused on the experiences of females. Ten studies included females aged between 15 and 49 years –40, threTwenty-one journal articles, two policy submissions , 46, andSixteen studies specified their data collection methods. Eight conducted interviews including semi-structured , 44, 48,Three studies specified their sample eligibility criteria , 51, 53.At the most basic level reproductive coercion can be understood as anything that may impact reproductive choices and autonomy. Most research defines reproductive coercion as ‘behaviour’ that occurs at the interpersonal level  ‘interfering’ with women’s reproductive ‘autonomy’ through acts such as contraceptive ‘sabotage’, ‘coercion’ to get pregnant, or ‘controlling’ the outcome of a pregnancy. Two articles , 47 specThematic analysis identified four main themes which describe the ways in which reproductive coercions is defined: Individual external exertion of control over a woman’s reproductive autonomy; Systems and structures; Social and cultural determinants; Freedom from external forces to achieve reproductive autonomy. The relation between these themes is depicted in Fig. Individual external exertion of control over a woman’s reproductive autonomy captured the interpersonal level of reproductive coercion. Most definitions of reproductive coercion (16 articles) were positioned at the interpersonal level [The theme al level , 51, 53.Within this frame, reproductive coercion is contextualised as a form of intimate partner violence, usually perpetrated by a man against a woman, with reproductive coercion just ‘one type of coercive behaviour used by some abusers’ (p248). AThe definitions that focus on the interpersonal level use words such as ‘control’ , 39, 48,These definitions suggest the goal of reproductive coercion is to ‘purposefully’  ‘interfeSystems and structures describes organisational, institutional, structural and systems level forms of reproductive coercion and relates to Freedom from external forces to achieve reproductive autonomy. Ten of the articles in this review [Individual external exertion of control over a woman’s reproductive autonomy) to acknowledge that reproductive coercion can also be perpetrated by ‘organised criminal activity’ [Systems and structures can also be ‘protective factors’ which contribute to obtaining reproductive autonomy free from coercion [The theme s review , 52, 53 ctivity’ , healthcctivity’ , 41, andctivity’ , 49, 52.ctivity’ . These ictivity’ , are desctivity’ . In doinctivity’ (p5). Concoercion .These understandings of reproductive coercion highlight that it is ‘bidirectional’, a ‘spectrum’ or continuum from ‘subtle to overt’, not a dichotomy, and that reproductive coercion is a ‘structural phenomenon rather than simply an interpersonal one’ (p8). ‘PoWords such as ‘power’ to ‘establish’ and ‘maintain’ ‘control’ of women’s reproduction are used in these definitions, particularly in reference to already marginalised and othered populations  highlighting the intersections of ‘multiple layers of structural oppression’ (p2). WorThese explanations of reproductive coercion draw attention to the inequities experienced by marginalised and othered populations which are exacerbated by ‘anti-choice reproductive health policies’, and can further reduce access to reproductive health services (p36). FoWithin healthcare institutions which are governed by not only their own policies but also the policies, laws, and legislation of the state, reproductive coercion is reproduced and enacted due to their coercive effect. This is also apparent in healthcare provider actions which reflect the coercive policies of the institution and state. For example, ‘downward coercion’ whereby ‘accessible and affordable contraception’ required for reproductive autonomy is not available and simultaneously ‘upward coercion’ (p3) whenThese descriptions of reproductive coercion suggest the goal is to give more attention to the ‘structural’ in addition to the ‘individual’ form of coercion, recognising that reproductive coercion is a continuum. Further, to better understand the ‘everyday ways that women’ (p3) see Social and cultural determinants which highlights the social and cultural conditions and contexts, including concepts of gender and cultural norms, and discrimination primarily driven by dominant ideologies and discourses including racism and patriarchy. Here it becomes apparent that denial of reproductive autonomy through coercive practices is shaped by ‘misogyny’, ‘white supremacy’ and ‘neoliberalism’ [Systems and structures, this theme necessitates the need for an intersectional lens in definitions of reproductive coercion. This theme demonstrates how reproductive coercion which manifests at the interpersonal level  is shaped by the ideologies underpinning and constituting reproductive coercion, and the social and cultural norms which act as ‘contributing factors’ to limit women’s autonomy [Nine articles −49, 52 cautonomy .Social and cultural understandings and practices ‘interfere with some of the most fundamental aspects of a woman’s reproductive autonomy and bodily integrity’ (p3) and Social and cultural norms and values regarding women’s role in society, ‘pregnancy, motherhood, and reproduction’ (p1403) nThe conditions in which a woman lives and experiences her everyday world provides the context in which reproductive decisions are made. While not necessarily direct coercive practices, these underlying conditions create indirect coercive control of a woman’s reproductive autonomy. For example, lack of access due to either resources  or structures, transport and healthcare institutions / providers, education, housing, social exclusion, or residency or citizenship status which may afford full or restricted rights and access to women , 33, shaFreedom from external forces to achieve reproductive autonomy, captured the broader reproductive rights, justice, and autonomy lens identified in four of the included articles [The final theme, articles , 43, 52,articles (p157) anIndividual external exertion of control over a woman’s reproductive autonomy, to more positive and empowering language. Further, these definitions tend to be underpinned by human rights, which include reproductive rights, for example, the right to free choice to have a child or not, and the right to have a child ‘in a safe and healthy environment which is free from individual and state violence’ [These explanations of reproductive coercion use words such as ‘freedoms’, ‘human rights’, ‘rights-based approach’, ‘gender-sensitive’, and ‘reproductive autonomy’, shifting away from the negative language of the definitions described in the theme iolence’ (p2), andiolence’ (p12). ThThese descriptions of reproductive coercion suggest the goal is to be inclusive of the intersectionalities of women’s lives and their position in society to demonstrate that choice is constrained, and that there is ‘a duty to respect and protect individual choice in matters relating to reproduction’ which ‘requires the state to take steps to render the choice meaningful for realizing reproductive health’ for example, through the ‘provision of means to exercise choice’ (p129).The findings of this scoping review reveal most definitions of reproductive coercion are situated within the interpersonal level, ignoring the interconnected structural, social, and cultural factors that create and perpetuate women’s reproductive coercion. This has led to a narrow definition of reproductive coercion, which does not take account of the individual within their broader setting. Defining reproductive coercion at the interpersonal level has been an important starting point in getting this public health issue recognised and on the broader health priority agenda. However, we now need to move beyond this conceptualisation to take account of the inter-related multi-level factors. Some articles included in this scoping review contend that the broader conditions beyond the interpersonal level contribute to reproductive coercion but do not constitute reproductive coercion, and thus should not be considered as part of the definition of the concept , 47. HowSocial and cultural determinants shape the constitution and practices of organisations, institutions and systems discussed in theme Systems and structures. These institutions and systems in-turn create the operational conditions including policies and services in which reproductive rights, justice and autonomy are enabled and enacted (or not). Through explanations of reproductive coercion, using a reproductive justice lens, ‘intersectionality and the language of human rights to address the power asymmetries that arise due to citizenship, gender, race, caste, ethnicity, class, abilities’ [The analysis revealed the themes operate in an inter-related way rather than being independent, emphasising and reifying the influence of the structural and systemic contexts of reproductive coercion. For instance, dominant societal ideologies and discourses discussed in theme ilities’ (p4) are Systems and structures in regards to ‘incentivizing’, commercial reproductive coercion was not explicit in any of the identified research included in this review. Commercial reproductive coercion may be perpetrated by healthcare providers whereby women feel coerced into agreeing to additional or more expensive treatments through fear or pressure. For example, women/couples seeking in vitro fertilisation (IVF) to conceive may feel pressured to sign up for additional treatments in fear of not conceiving if they do not. These IVF add-ons are procedures or medicines claiming to increase the chance of a pregnancy, mostly in countries where IVF is delivered in a commercial setting, including the USA and Australia [Missing from the research identified in this scoping review is reproductive coercion by commercial entities or interests. While this was implicitly discussed in ustralia . While wustralia .As with all scoping reviews, this review had its limitations. While scoping reviews do not formally appraise the quality of the evidence in the studies reviewed , the artThough a comprehensive and systematic search strategy was designed and undertaken by the research team, studies of relevance to the aim of this review may have been missed. As a result, the findings are a reflection of the analysis of the articles included in this review. However, the strengths of this review were the systematic approach taken and the use of multiple reviewers in the screening of title and abstracts and full-texts to ensure strict application of the eligibility criteria. Further, the review reported on a range of study types. Finally, this scoping review has provided important insights into the current state of how reproductive coercion is defined.The aim of this scoping review was to explore how reproductive coercion is defined, how it is defined at the social-cultural-systems-structural levels, and if the definitions of reproductive coercion are inclusive of the conditions and contexts in which reproductive coercion occurs. The identification of four interacting themes on current definitions of reproductive coercion suggests four key findings: (1) reproductive coercion is experienced in multiple ways within and across a continuum from the interpersonal to the systems, structural and societal; (2) is exercised at multiple interacting levels in an effort to diminish reproductive decision-making freedoms and choices by controlling or limiting access to reproductive options through policy, law, legislation, behaviours, attitudes and so forth; (3) is socially and culturally embedded systematic control and oppression of females reproductive rights enacted across multiple levels to hold power over female’s reproductive autonomy creating, maintaining, and reinforcing gender inequality and other intersectional forms of oppression ; and (4) manifests at the interpersonal level as an artifact of intersecting social, cultural, institutional systems and structures, and organisations and the state which set the necessary context within which reproductive coercion occurs.We argue for a more inclusive definition of reproductive coercion that recognises the gendered nature and female’s multiple oppression and inequality. Further, reproductive coercion is, and can be, perpetrated and/or facilitated by the state and that it is produced and enacted at the societal, institutional, systems, and structural levels. We propose the following definition of reproductive coercion to capture the multiple interacting layers within which reproductive coercion occurs:Reproductive coercion is the act of removing or limiting reproductive autonomy to control reproductive decision-making freedoms and choices to hold power over reproductive autonomy. It occurs through socially and culturally embedded systematic control and oppression of reproductive rights, beliefs, conceptualisations of gender roles, behaviours, attitudes, and actions, practices, policy, law, and legislation resulting in gender inequality and other intersecting forms of oppression . This manifests and is experienced at the interpersonal level in multiple ways as an artifact of interconnected and interacting forces across the social, cultural, institutional systems and structures, organisations, and the state which create the context within which reproductive coercion occurs.This definition is explicitly inclusive of the broader conditions and context in which reproductive coercion manifests which is required to effectively implement settings based, whole-of-population, primary prevention programs using an intersectional approach to reduce reproductive coercion and the subsequent negative health outcomes. Further research is required to explore how the ways in which reproductive coercion is defined impacts public health primary prevention approaches and outcomes, including if adopting a broader more inclusive definition of reproductive coercion would facilitate improved reproductive autonomy. This scoping review and the resulting definition have provided the important first step in situating reproductive coercion beyond the interpersonal level to allow for the primary prevention of this important public health issue.Below is the link to the electronic supplementary material.Supplementary Material 1


Reproductive function is critical for species survival; however, it is energetically costly and physically demanding. Reproductive suppression is therefore a physiologically appropriate adaptation to certain ecological, environmental, and/or temporal conditions. This ‘allostatic’ suppression of fertility enables individuals to accommodate unfavorable reproductive circumstances and safeguard survival. The mechanisms underpinning this reproductive suppression are complex, yet culminate with the reduced secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, which in turn suppresses gonadotropin release from the pituitary, thereby impairing gonadal function. The focus of this review will be on the role of RFamide-related peptide (RFRP) neurons in different examples of allostatic reproductive suppression. RFRP neurons release the RFRP-3 peptide, which negatively regulates GnRH neurons and thus appears to act as a ‘brake’ on the neuroendocrine reproductive axis. In a multitude of predictable  and unpredictable  situations in which GnRH secretion is suppressed, the RFRP neurons have been suggested to act as modulators. This review examines evidence for and against these roles. Reproductive function is fundamental for species survival. However, it is physiologically costly—especially in mammals—due to the high energetic demands of pregnancy, lactation and rearing of offspring. When environmental or physiological conditions are not conducive for reproduction, neuroendocrine pathways that can exert allostatic control over reproductive function cause acute reproductive suppression. This ‘allostatic’ reproductive suppression is adaptive, as it enables individuals to accommodate unfavorable changes in their environment and/or in their physiology in order to safeguard survival. According to McEwen and Wingfield , allostaThere are many examples of allostatic reproductive suppression. For example, in seasonal breeders, reproductive suppression is predictable, such that fertility is coordinated to ensure offspring are born when environmental conditions are most favorable for survival. During different life cycle stages, such as pre-puberty and post-menopause, reproductive function is also predictably suppressed. However, unpredictable events can also cause reproductive suppression, such as certain environmental and/or physiological states. During times of severe metabolic stress, for example due to famine, energy demand exceeds energy supply, which will initiate an allostatic response that directs an individual away from energetically costly breeding and into survival mode. During times of psychosocial trauma or severe illness, which are also unfavorable times to reproduce, sufficient energy may be present, but the activation of the stress systems can induce a facultative physiological and/or behavioral response that ultimately defers reproduction ,2. AllosTo understand the mechanisms underpinning allostatic reproductive suppression, it is necessary to first understand how the reproductive axis—the hypothalamo-pituitary-gonadal (HPG) axis—is regulated. The hypothalamic gonadotropin-releasing hormone (GnRH) neurons governing the HPG axis serve as the final integrators of a multitude of neuroendocrine and hormonal inputs . GnRH redecrease in RFRP neuronal activity is the preovulatory GnRH/LH surge [Rfrp gene expression, RFRP cell numbers, and RFRP + cFos coexpression  all decline on the afternoon of the surge in naturally cycling or surge-induced hamsters and mice [RFamides form a class of neuropeptides characterized by an arginine–phenylalanine–amide motif at their C terminus. They were first identified in 1977 in molluscan cerebral ganglia, but almost another decade passed before they were purified and chemically characterized in any mammalian species. There are now five RFamide families known to exist in the mammalian central nervous system, where their widespread fiber distribution implies varied roles including regulation of energy balance, behavior, and reproduction . The RFRLH surge . Hypothaand mice ,26. Thisand mice ,4.As mentioned previously, RFRP neurons appear to be involved in both the temporal regulation of reproductive function, which causes predictable cycles of reproductive activation and inactivation, as well as in stress-related reproductive regulation, which allows for facultative reproductive suppression. RFRP neurons are thus emerging as important integrators of environmental and physiological cues within the neuroendocrine reproductive axis, such that they release RFRP-3 peptide to act as a ‘gate’ or ‘brake’ on reproductive function when the afferent inputs they receive suggest conditions are not optimal see . HoweverMany studies have explored the impact(s) of exogenous central RFRP-3 administration on reproductive function. In contrast to the variable results obtained from peripheral administration, these studies mostly support the concept that RFRP-3 acts as a brake on GnRH-induced LH release and reproductive drive. For example, intracerebroventicular (ICV) injection of RFRP-3 to male rats significantly suppressed all facets of male sexual behavior and also reduced plasma levels of LH . ICV RFRRfrp gene silencing using targeted shRNA alleviated various stress-induced infertility measures in female rats [Rfrp, thereby inhibiting its effect, show elevated gonadotropin levels [To further investigate the in vivo role of RFRP-3 on the HPG axis, our lab generated a novel RFRP-Cre mouse line, which we then crossed with Cre-dependent designer receptor (DREADD)- or diphtheria toxin receptor-expressing mice to investigate the effects of endogenous RFRP-3 excitation and inhibition, as well as RFRP neuronal ablation . Chronicale rats , suggestale rats  . WhiSeasonal breeding is crucial to the survival and reproductive success of the vast majority of vertebrate species inhabiting temperate regions. Seasonal changes in environmental factors, such as photoperiod, lead to alternating periods of photosensitivity and photorefractoriness in small birds and short-lived mammals to bring about alternating periods of reproductive activity and quiescence, the latter being maintained by an increased potency of negative feedback regulation. In larger long-lived animals, the environmental cues interact with the underlying endogenous rhythm, producing the same reproductive outcome. The timing of reproductive activity, which precedes the birth period by the duration of gestation, ensures that the young are born during a season in which the environmental conditions most favor neonatal survival. Photoperiodic cues can act directly on photoreceptors on the GnRH neuronal network in birds, whereas in mammals they are encoded and communicated to reproductive (and other) brain control centers in the form of elevated secretion of the pineal hormone melatonin during the hours of darkness, which are extended in duration during winter and shorter in summer in temperate regions ,50.It would be reasonable to hypothesize that RFRP neuronal activity would be higher during reproductive quiescence than during the breeding season. Consistent with this hypothesis, more RFRP-3 immunoreactive cell bodies were observed during the non-breeding season  than the breeding season in male macaques  . SimilarRfrp mRNA and immunoreactive RFRP neurons compared to virgin diestrus controls, and this effect was attributable to the presence of elevated circulating prolactin concentrations in the lactating animals . In another study that employed a different experimental design (lactating rats with pups removed were used as controls), the suckling stimulus exerted the opposite effect: it increased Rfrp mRNA levels, and the authors suggested this might be a cause of lactational infertility [Rfrp expression.There are currently very few studies in which the role of RFRP-3 in lactational infertility has been tested. Conceptually, the output of RFRP neurons might increase to cause lactational amenorrhea since they can inhibit reproductive function. Alternatively, it might decrease since lactation is associated with stress hyporesponsiveness  and RFRPertility . ClearlyStress-related reproductive dysfunction is perhaps the most notable form of allostatic reproductive suppression. The reproductive impairments can be subtle, such as reduced hormone secretion, or severe, such as the complete suppression of puberty onset and/or fertility . While mRfrp gene expression in vitro [Rfrp gene expression in male and female rats [Rfrp gene silencing using a targeted shRNA prevented stress-induced infertility in female rats [Importantly, approximately half of RFRP neurons express GR , and corin vitro . Physiolale rats ,67, but ale rats . Data frale rats . Similarale rats . A singlale rats , indicatRfrp mRNA levels [Metabolic challenges, such as starvation, inhibit fertility and sexual behaviors. Many of the neurons that provide important input to the reproductive axis are also involved in control of metabolic functions, and this allows the availability of metabolic fuels to be coordinated with fertility . For exaA levels  or no deA levels  relativeA levels . TogetheIn Syrian hamsters, the percent of RFRP cells co-expressing c-Fos  was significantly increased at 8 and 12 days after food restriction . The effRfrp and Npffr1 mRNA levels, and these were negatively correlated with Gnrh mRNA and serum LH levels [Rfrp mRNA despite causing a reduction in serum LH levels [Immune stress, such as infection and inflammation, can also have a negative impact on fertility ,78,79. EH levels . In anotH levels . HoweverH levels , suggestH levels ,67,69, bRfrp mRNA, which correlates with a downregulation of serum LH in male rats [Rfrp mRNA, since it was prevented by adrenalectomy [Rfrp expression associated with suppressed sexual maturation in non-dominant naked mole rats, RFRP-3 administration was sufficient to sustain their suppressed sexual maturation, even when the individuals were removed from the dominance cues [As with metabolic and immune stress, which are physiological stressors, psychosocial stress  can likewise cause allostatic reproductive suppression. However, unlike metabolic and immune stress, which directly threaten an individual’s survival, psychosocial stress is purely a perceived threat. In humans, depression and other mood disorders are examples of psychosocial stress associated with reproductive dysfunction . One comale rats . GC relealectomy . We havealectomy . Furtheralectomy ,31,83,84alectomy  and othealectomy . Not onlnce cues . CollectThe mechanisms underpinning adaptive reproductive suppression are complex and involve a multitude of factors. The RFRP-3 neuropeptide system appears to play a modulatory role in integrating many of these factors and acting as a ‘brake’ on reproductive drive when conditions are unfavorable. RFRP neurons appear to contribute to stress-induced and (in some species) seasonal reproductive suppression, while their role in mediating changes in reproductive competency during different life cycle stages  is less clear and would benefit from functional data from manipulating RFRP neuronal activity see . With th


BRCA1/2-mutant (BRCAMUT) TNBC, durable responses will likely be achieved if PARPi are used in combination. It is plausible that sequential administration of a potent PARPi like talazoparib in combination with carboplatin can enhance primary tumour and metastasis inhibition in BRCAMUT and BRCA1/2 wild-type (BRCAWT) TNBCs, and decrease toxicity.Patients with triple-negative breast cancer (TNBC) develop early recurrence. While PARP inhibitors (PARPi) have demonstrated potential in We evaluated the impact of the concurrent combination of talazoparib and carboplatin on cell survival in 13 TNBC cell lines. We compared the concurrent and sequential combination upon fork replication, migration and invasion. We also used three orthotopic xenograft models to evaluate primary tumour growth, distant metastasis, and toxicity.BRCA1/2-mutation status. The sequential combination decreased fork speed in normal cells, but not in TNBC cells. The talazoparib-first sequential combination resulted in a strong reduction in migration , invasion , lung micrometastasis , and less toxicity in a BRCAWT model.Concurrent talazoparib and carboplatin was synergistic in 92.3% of TNBC cell lines, independent of The sequential combination of talazoparib and carboplatin is an effective approach to inhibit micrometastatic disease, providing rationale for the use of this combination in early TNBC patients. BRCA1/2 (gBRCAMUT). However, patients with gBRCAMUT constitute about 11–20% of all TNBCs   and Mouse anti-BrdU [clone B44] antibodies , overnight at 4 °C. Slides were incubated with anti-Rat AlexaFluor 568  and anti-Mouse AlexaFluor 488  antibodies, for 1 h at room temperature. Slides were mounted in ProLong™ Gold antifade Mountant  and analysed using a Zeiss Axio Observer Z1 microscope with ×63 oil objective. Replication fork speed was calculated using the CldU + IdU track length/60 min * 2.59 kb/µm.Cells were sequentially labelled with 25 µM of 5-Chloro-2′-deoxyuridine (CldU)  and then 250 µM of 5-Iodo-2’-deoxyuridine (IdU)  for 30 min each . Cells w3, mice were randomised into treatment groups based on tumour volume volumes and weight. Our sample size was based on the experiments previously described [All animal experiments were approved by the Institutional Animal Protection Committee (CIPA) of the Centre de Recherche de Centre hospitalier de l’Université de Montréal (CRCHUM) under protocol C17017SHs. Either 5 million MDAMB231 or 2 million MX1/HCC1806 cells were resuspended in 50% Matrigel Matrix Phenol Red Free , 25% PBS and 25% collagen type 1  solution . Using 74 or 5 × 104 cells were suspended in serum-free media in 8-μm transwell inserts , for 24-h migration or 48-h invasion assays, respectively. For the invasion assay, the upper chamber was pre-coated with 1/9 dilution of Matrigel . After incubation, inserts were washed with PBS. Migrated or invaded cells on the underside of the membrane were fixed with 4% paraformaldehyde and stained with hematoxylin . Migrated or invaded cells were counted from six representative fields from the inverted EVOS XL core microscope (Thermo Fisher Scientific).In all, 3 × 10In total, 200 μg of protein was extracted from cell lysates from MDAMB231 cells to detect the expression of 31 chemokines using the human chemokine array kit . Images of membranes were acquired with ChemiDoc (BioRad) and dots were quantified using QuickSpots (Western vision) software. At the same time point, supernatants of MDAMB231 and HCC1395 cells were collected to detect MCP-1 levels with the human CCL2/MCP-1 Immunoassay .See Supplementary Methods for details regarding RNA-seq analysis.P < 0.05 is considered statistically significant.Normality of each dataset was first verified before performing Kruskal–Wallis ANOVA or one-way ordinal ANOVA with multiple-comparison post test. Two-way ANOVAs with post-tests were performed for grouped conditions. Data are represented as mean + /− SEM, except for fork speed, in which median values with interquartile ranges are presented. Data were analysed and plotted using GraphPad Prism software 8. Further information on research design is available in the 50 values of talazoparib and carboplatin as single agents in a panel of 13 TNBC cell lines  values <1 in 92.3% (12/13) of cell lines  were calculated to determine the dose-fold reduction of each therapeutic agent to achieve synergy. DRI for talazoparib ranged from 1.5 to 3.8, while the DRI for carboplatin ranged from 2.1 to 7.1. This is suggestive that synergy can be accomplished with important dose reductions of both drugs, with greater reductions in carboplatin versus talazoparib commonly observed in 61.5% (8/13) of cell lines.We determined the IC μM Fig. . Using tnes Fig. . Stronge+ )  of talazoparib (T1-9), carboplatin (C1-9), or the concomitant combination of talazoparib and carboplatin (TC1-9) upon 53BP1 response and apoptosis using a 10-day chemosensitivity assay in 14 TNBC cell lines Fig. . DNA dam+ ) Fig. . While cWT cells, including two PARPi-resistant cell lines, HCC1143 and MDAMB231, a PARPi-sensitive yet carboplatin-resistant cell line, HCC1806; and one PARPi-sensitive cell line that is both BRCA1-deleted and BRCA2-mutated, MX1. In all four cell lines, cell survival was comparable between the concomitant and sequential combination approaches, with either talazoparib or olaparib as the PARPi backbone  and two sequential approaches: carboplatin first followed by talazoparib (seq.C→T), and talazoparib run-in approach followed by carboplatin (seq.T→C) upon cell survival using the 10-day chemosensitivity assay Fig. . We seleone Fig. .Fig. 3DiTo better understand the impact of the combination approaches on in vitro toxicity, we compared fork replication speed and DNA damage response between TNBC and normal cells. We used two TNBC cell lines, HCC1806 and MDAMB231, and two human normal cell lines including MCF10A  and BM-MSC  Fig. . We deteP < 0.0001), 48.9% (P < 0.0001), 59.2% (P < 0.0001), and 59.9% (P < 0.0001) in MDAMB231, HCC1806, MCF10A and BM-MSC cells, respectively  and 14.8% (P = 0.0005) reduction in median fork speed in comparison to the concomitant combination, in MCF10A and BM-MSC cells, respectively. Conc.T + C resulted in the highest proportion of γ-H2AX+ cells in MCF10A  and BM-MSC  in comparison to control , in comparison to conc.T + C. We also evaluated apoptosis in MCF10A and BM-MSC cells , and 31.7% (P < 0.0001) for MCF10A and BM-MSC, respectively.However, in normal cells, sequential treatments decreased fork speed in comparison to concomitant treatments. Seq.T→C demonstrated a 10.3% , 75.5% (P = 0.0001), 71.7% (P = 0.002), by the conc.T + C., seq.C→T, and seq.T→C strategies, respectively, in comparison to control  with conc. T + C, 46.6% (P = 0.04) with seq.C→T, and 62.0% with seq.T→C (P < 0.0001) in comparison to control , and 66.7% (P < 0.0001) reductions in tumour volume, respectively, which was more remarkable than the 51.4% reduction (P = 0.08) with conc.T + C, in comparison to control , while seq.C→T demonstrated the greatest gain in weight at 10.4%. In the HCC1806 cohort  Fig. .P = 0.002), 1.5 (P = 0.0003), and 1.7 (P = 0.0098), for conc.T + C, seq.C→T, and seq.T→C respectively, in comparison to control (mean 4.9) , seq.C→T  and seq.T→C , in comparison to control (mean 4.3)  Fig. . Similar.3) Fig. . No stat.3) Fig. .No changes in haemoglobin (Hgb) levels were observed with treatment in the MX1 cohort Fig. . While tP = 0.003) and seq.T→C led to a 56.4% (P < 0.0001) reduction in lung micrometastases, which was not observed with the concomitant combination  , and 36.9% in comparison to the concomitant approach (P < 0.05). Similar trends were also observed with liver micrometastasis  reduction in micrometastasis, which was similar to the 76.3% (P = 0.02) reduction with the seq. T→C combination.To further evaluate the impact of the combination of talazoparib and carboplatin on distant metastasis, we used an orthotopic xenograft model of MDAMB231, which is known for its high metastatic efficiency . In compNS) Fig. . The seqsis Fig. . In compP = 0.05) and 30.8% (P = 0.01) reduction in cell migration, respectively. Once again, the seq.T→C demonstrated the most striking inhibition in cell migration  reduction in comparison to control, 61.1% reduction (P < 0.0001) in comparison to talazoparib, and 39.0% reduction (P = 0.008) in comparison to conc.T + C. The other combination strategies also inhibited migration with 51.5% (P < 0.0001) reduction for the concomitant approach, and 38.9% (P < 0.0001) for the seq.C→T approach. The treatments as single agents or in combination demonstrated a similar trend with HS578T  reduction in cell migration.To further dissect the metastatic process, we evaluated the impact of talazoparib as monotherapy and in combination on cell migration and invasion in vitro Fig. . In MDAMion Fig. , with 70P < 0.0001), 47.0% (P < 0.0001), 47.5% (P < 0.0001), and 41.3% (P = 0.0005) reduction in comparison to control, talazoparib, carboplatin, seq.C→T, and conc.T + C, respectively  inhibition in cell invasion in comparison to control. In HS578T, the greatest inhibition was also demonstrated by seq.T→C with a 26.7% (P < 0.0001), 18.9% (P = 0.0002), and 21.3% (P < 0.0001) reduction in cell invasion in comparison to control, talazoparib, and carboplatin, respectively  reduction in cell invasion, in comparison to control. Similar results were obtained with other PARPi. Olaparib or niraparib resulted in the greatest reduction in migration or invasion when administered witih the sequential PARPi-first combination approach , 30.7% reduction in comparison to carboplatin (P < 0.0001), and a 11.8% reduction in comparison to conc.T + C (P = 0.03). In HCC1395, seq.T→C also reduced MCP-1 levels by 31.5% in comparison to control (P < 0.0001), and by 33.9% in comparison to carboplatin (P < 0.0001). Conc.T + C also reduced MCP-1 levels by 19.5% (P = 0.049), and seq.C→T reduced MCP-1 levels by 21.7% (P = 0.01). Since the greatest downregulation of MCP-1 levels was demonstration by seq. T→C in comparison to carboplatin, this is suggestive that the sequential combination of talazoparib and carboplatin may be an important approach to inhibit MCP-1 levels that may otherwise be upregulated with carboplatin.CCL2/MCP (Monocyte chemoattractant protein)-1 drives migration and invasion, and is implicated in cancer cell homing at the metastatic site , 39. PARTo better understand the underlying mechanisms for enhanced inhibition of metastasis, we also performed gene expression analysis of the metastatic lung tissue of the MDAMB231 xenograft to determine if there are differentially expressed pathways in the human cancer tissue between the treatment groups (Supplementary Figs. Altogether, the sequential administration of talazoparib and carboplatin resulted in a noteworthy reduction in distant metastasis, cell migration and invasion. Our results are suggestive that the PARPi-first combination may have a distinct mechanism of action in comparison to other combination approaches, which can significantly enhance the efficacy of each therapeutic agent.MUT models [MUT and BRCAWT subtypes. Talazoparib and carboplatin demonstrated a mean dose-reduction index of 2.8- and 3.7-fold, respectively. Similarly, sustained DNA damage and apoptosis responses were observed at lower drug concentrations of the combination, particularly in the PARPi-resistant cell lines. These results could partly explain the toxicity observed with the concomitant combination of talazoparib and carboplatin in a phase I trial, in which only a 0.25-fold reduction in talazoparib and carboplatin doses were utilised [Preclinical studies have commonly focused on the combination of PARPi and platinum-based therapy using BRCAT models , 44. To utilised .MUT model, MX1, the least weight gain and haematologic toxicity was demonstrated by the talazoparib-first approach in the BRCAWT model, MDAMB231. Since the same background strain was used, one plausible explanation for the differential toxicity could be different inflammatory responses induced by the BRCAMUT versus BRCAWT tumours [MUT context and talazoparib-first approach in the BRCAWT context may offer patients the greatest safety profiles.Sequential talazoparib and carboplatin was associated with a reduction in fork speed, DNA damage, and cell death in normal cells with, in comparison to TNBC cells. This is similar to what was previously reported: higher endogenous fork speeds correlated with lower endogenous replication stress in normal cells, which explained the reduced toxicity observed with the sequential PARPi combination approaches . In our  tumours , 46, whiFor our in vivo experiments, we based our dosing schedule of one dose of carboplatin and nine days of talazoparib on a xenograft model of subcutaneous tumour fragment implantation . Our resWT model with high metastatic efficiency. We showed comparable efficacy of the concurrent and sequential approaches for primary tumour inhibition. However, to our knowledge, we are the first to report that the sequential combination approaches strongly inhibit migration, invasion, and distant metastasis. The talazoparib-first combination demonstrated the greatest reduction in cell migration and invasion at 70.4% and 56.9%, respectively, with an enhanced effect in comparison to concurrent combination. In vivo, the talazoparib-first approach resulted in a 56.4% and 76.3% inhibition in lung and liver metastasis, respectively. Since we did not identify a differential impact on the tumour volumes with the sequential or concomitant combination approach, it is plausible that the impact upon migration and metastasis is independent of treatment effects on the primary tumour.While we understand some of the limitations of cells lines and their derived models , the strFurthermore, the talazoparib-first combination was associated with decreased expression of seven chemokines. The expression of one such chemokine, MCP-1, was downregulated the greatest with the talazoparib-first combination. Interestingly, chemotherapy including cisplatin has been shown to promote secretion of MCP-1, recruit inflammatory monocytes enriched with the receptor of MCP-1, CCR2, forming pre-metastatic niches, thus promoting distant metastasis in breast and lung cancer , 51. SucWT model.Finally, we evaluated the pathways that were differentially expressed by the different treatment groups using metastatic lung tissue. We found that DNA damage response pathways were distinctly downregulated with seq.T→C in comparison to control and other combination groups. We and others have previously shown that the downregulation of these pathways allow for enhanced sensitivity to PARPi , 52, sugTaken together, we have provided a comprehensive preclinical analysis of the combination of talazoparib and carboplatin in TNBC. We have demonstrated that the combination is synergistic in most TNBC cell lines. While the sequence of administration does not impact tumour proliferation or tumour growth, sequential administration of talazoparib and carboplatin can decrease toxicity and significantly inhibit migration, invasion, and distant metastasis. Therefore, our results lead the way to future clinical trials with the evaluation of the sequential combination of talazoparib and carboplatin in early breast cancer, potentially providing an effective approach to eradicate micrometastatic disease in TNBC patients.Supplementary MethodsSupplementary FiguresSupplementary Table 1ARRIVE ChecklistReporting Summary


Metastases are the primary cause of morbidity and mortality in cancer patients, and solid tumour metastases are mostly located in lymph nodes. Currently, there are no techniques that model the pre-metastatic lymph node microenvironment in vitro. In this study, we prepared a novel extracellular matrix, Lymphogel, which is derived from lymph nodes, mimicking the tumour microenvironment (TME) of metastatic carcinoma cells. We tested the suitability of the new matrix in various functional experiments and compared the results with those obtained using existing matrices.We used both commercial and patient-derived primary and metastatic oral tongue squamous cell carcinoma (OTSCC) cell lines. We characterized the functional differences of these cells using three different matrices , porcine normal neck lymph node-derived Lymphogel (p-LG) in proliferation, adhesion, migration and invasion assays. We also performed proteomic analyses to compare the different matrices in relation to their functional properties.OTSCC cells exhibited different adhesion and invasion patterns depending on the matrix. Metastatic cell lines showed improved ability to adhere to h-LG, but the effects of the matrices on cell invasion fluctuated non-significantly between the cell lines. Proteomic analyses showed that the protein composition between matrices was highly variable; Myogel contained 618, p-LG 1823 and h-LG 1520 different proteins. The comparison of all three matrices revealed only 120 common proteins. Analysis of cellular pathways and processes associated with proteomes of each matrix revealed similarities of Myogel with h-LG but less with p-LG. Similarly, p-LG contained the least adhesion-related proteins compared with Myogel and h-LG. The highest number of unique adhesion-related proteins was present in h-LG.We demonstrated that human pre-metastatic neck lymph node-derived matrix is suitable for studying metastatic OTSCC cells. As a whole-protein extract, h-LG provides new opportunities for in vitro carcinoma cell culture experiments.The online version contains supplementary material available at 10.1186/s12885-023-11275-6. Although 2D culture settings on plastic are simple and convenient to use, they have many critical limitations when aiming to replicate the complexity and pathophysiology of a tumour. Under such simplified conditions, tissue-specific architecture, mechanical and biochemical signals and cell-cell and cell-matrix interactions are lost . Cell adCancer research, including drug testing, has traditionally focused mostly on cancer cells originating from primary tumours. However, in many solid cancers, metastases are the main cause of cancer deaths, and the availability of efficient drugs to prevent or treat metastatic tumours is very limited . DespiteWe formerly developed the first human tumour-derived matrix, Myogel . It is aOf head and neck cancers, oral tongue squamous cell carcinoma (OTSCC) is the most prevalent cancer in the oral cavity, and despite improvements in treatment, it remains characterized by poor survival, mostly because of its tendency for LN metastasis . HoweverThe experimental protocol for use of OTSCC patient samples was approved by the Ethics Committee of the Northern Ostrobothnia Hospital District, Finland (statement no. 31/2016). Four primary  and three metastatic  tumour cell lines were isolated from OTSCC patient samples at Oulu University Hospital after informed consent. Clinical characteristics of OU-OTSCC cell lines are described in Additional file 1: Table 2 and 95% humidity. All the cell lines used were at passage number under 30.UT-SCC-24A , UT-SCC-24B , UT-SCC-42A  and UT-SCC-42B  have been established at the Department of Otorhinolaryngology – Head and Neck Surgery, Turku University Hospital, Finland and were kindly provided by Professor Reidar Grénman. UT-SCC and the commercial human OTSCC HSC-3 (Japanese Collection of Research Bioresources (JCRB) Cell Bank, JCRB0623) cell lines were cultured in DMEM/F-12 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin , 0.4 µg/ml hydrocortisone (Merck), 50 µg/ml ascorbic acid  and 250 ng/ml amphotericin B (Merck). All cell lines were maintained at 37 °C with 5% COExperimental protocols on human leiomyoma tissue were approved by the Ethics Committee of the Northern Ostrobothnia Hospital District, Finland (statement no. 2/2017). Clinically, non-metastatic human cervical lymph node tissues  were collected from three oral or hypopharyngeal cancer patients undergoing neck dissection surgery. Based on the clinical evaluation, the patients had more than two metastases. The collected fresh tissue was evaluated to be macroscopically non-metastatic by the expert pathologist. The study was approved by the Ethics Committee of the Northern Ostrobothnia Hospital District, Finland (statement no. 31/2016). All patient samples were collected after informed consent was obtained.Sus scrofa domesticus) sacrificed for other experimental study at the University of Oulu Laboratory Animal Centre KEKS . The animals were purchased for the experimental study from a contract breeder  providing pigs for Oulu Laboratory Animal Centre. The original experimental study protocol was reviewed and approved by the National Animal Experiment Board of Finland (ESAVI/6941/04.10.07/2016) and animals were sedated with midazolam 0.2 mg/kg  and ketamine 10 mg/kg  combination. Anesthesia was induced with fentanyl (50 µg)  and maintained with sevoflurane (1-1.5%)  and infusion of fentanyl and midazolam. After surgical operations and experiment, the animals were euthanized with pentobarbital-Na 60 mg/kg .Normal anterior cervical lymph nodes were dissected from healthy domestic pigs . The next day, the discs were transferred onto steel grids in 12-well plates with 1 ml of medium. Cells were cultured on top of the myoma disc for 18–20 days. The tissues were fixed in 4% neutral-buffered formalin, dehydrated and embedded in paraffin. Then 6 μm sections were prepared for immunohistochemistry with monoclonal pan-cytokeratin AE1/AE3 antibody  as described previously -fibrinopeptide B  in 50% acetonitrile/0.1% formic acid solution at a flow rate of 0.3 µl/min was applied every 30 s.The samples were run in ion mobility assisted in data-independent analysis mode (HDMSE), in a Synapt G2-S mass spectrometer (Waters), by alternating between low collision energy (6 V) and high collision energy ramp in the transfer compartment (20 to 45 V) and using a 1-sec cycle time. The separated peptides were detected online with a mass spectrometer, operated in positive resolution mode in the range of Protein identification relative quantification between samples using precursor ion intensities was performed with Progenesis QI for Proteomics™ Informatics for Proteomics software . MSE parameters were set as follows: low energy threshold of 135 counts, elevated energy threshold of 30 counts, and intensity threshold of precursor/fragment ion cluster 750 counts.Database searches were carried out against Human Proteome UniProtKB-SwissProt database  with Ion Accounting algorithm and using the following parameters: peptide and fragment tolerance: automatic. Maximum protein mass: 750 kDa. Minimum fragment ions matches per protein ≥ 7. Minimum fragment ions matches per peptide ≥ 3. Minimum peptide matches per protein ≥ 1. Primary digest reagent: trypsin. Missed cleavages allowed: 2. Fixed modification: carbamidomethylation C. Variable modifications: deamidation of NQ residues. Oxidation of Methionine and false discovery rate (FDR) < 4%.Statistical analyses were carried out with SPSS Statistics 27 . To determine the statistical significance, we performed with one-way analysis of variance (ANOVA) followed by Bonferroni correction or independent samples T-test. P-value < 0.05 was considered statistically significant. Figures were created with Origin 2020 graphing software  and Adobe Photoshop CC 2019 .https://metascape.org/) and can be used to assess e.g., enrichment of biological and cellular pathways, drug targets and diseases of a user-provided list of genes/proteins. The total proteomes of each matrix were compared to identify common and unique proteins.Three technical replicates for each matrix were produced by proteomic analysis. To study the proteomes of each matrix, the data from replicates were combined, and duplicates were removed based on the protein accession number. Next, uncharacterized proteins along with proteins with duplicated descriptions (Names) were removed for each matrix. Proteins found in each matrix were analysed with Metascape  to identThe proteins (by protein entry) and intensities (by matched product) identified by proteomic analysis for three technical replicates of Myogel and h-LG were combined. For quality control and to compare levels of proteins present in both matrices, the proteins present in only one technical replicate or in only one matrix were filtered out. Missing single values were replaced by the average intensity of the two other technical replicates. The intensity values for each protein entry were log2-transformed and normalized with quantile normalization method. The mean transformed and normalized intensity values for each protein entry were compared between matrices, and a fold-change of 2 was used as the threshold to identify differentially expressed proteins between matrices. R version 4.2.2 with “dplyr”,”data.table” and ”zoo” packages were utilized for this analysis.Cell adhesion-related proteins common and unique to each matrix were extracted from the total proteome of each matrix by a comparison with proteins associated with the GO-term GO:0007155 (Cell Adhesion) in the GeneOntology (GO) database. Shared proteins and adhesion proteins with similar names and different accession numbers between human and porcine matrices were compared based on sequence identity on the BLAST tool from NCBI . Information on the cellular location for adhesion-related proteins were collected from UniProt via UniProt ID mapping tool .OU-OTSCC cell lines established from primary and metastatic tumours showed pancytokeratin immunostaining similar to HSC-3 cells, confirming that all cell lines are epithelial in origin and considered as carcinoma cell lines  and h-LG the largest (74) Fig. . Myogel Results of cancer progression, metastatic processes and drug testing may often be misleading due to the lack of biologically relevant study matrices. Particularly, a LN-derived matrix that would be suitable for metastatic cell lines remains unavailable on the market. In this study, we established three tongue cancer cell line pairs: OU-OTSCC-7, -9, -18 including primary tumour (A) and their corresponding lymph node metastases (B) cells. In addition, we established one primary tumour cell line, OU-SCC-15, and used a commercial human metastatic OTSCC cell line, HSC-3, as a control for aggressive tumour behaviour. We used these cell lines to assess the effect of three different TME matrices, human uterine leiomyoma-derived Myogel and normal porcine neck LN- and human HNSCC patients’ pre-metastatic neck LN-derived Lymphogels, on carcinoma cells’ proliferation, adhesion, migration and invasion. We also investigated the protein composition of Myogel and both Lymphogels and compared them with each other to elucidate the differences in cell behaviour.Primary and metastatic OTSCC cell lines showed similar cell viability on Myogel and both Lymphogels, confirming that all matrices had a protein composition that supports cell growth. In all primary-metastatic pairs, metastatic cells adhered more on human pre-metastatic LN-derived Lymphogel (h-LG). Although this difference between metastatic and primary cell adhesion on h-LG was clear, the same effect was not present on healthy porcine LN-derived Lymphogel (p-LG). When we studied OTSCC cells’ migration and invasion abilities on these different matrices by spheroid and wound healing assays, no clear matrix preference for primary or metastatic cell lines emerged. However, there was a non-significant trend that metastatic cells also invaded fastest on Myogel. This suggests that the protein composition, which induces the cell adhesion, may not support cell invasion. These results are in line with our previous findings that Myogel, compared with various other matrices, induces the invasion of cancer cells the most .We performed the pathway and process enrichment analysis by Metascape to study the most active pathways in each matrix. Common enriched clusters of functional characteristics in Myogel and h-LG were cytoskeleton-related pathways and processes. Processes unique to Myogel seemingly associated with ECM and cell adhesion such as ECM reorganization, matrix metalloproteases (MMPs) and Rho GTPases. MMPs and Rho GTPases are linked to numerous key cell functions such as cell cycle progression, cell migration, malignant transformation, cell polarity, invasion and metastasis , 29. In To focus on adhesion molecules for each matrix, a Gene Ontology functional annotation was applied to identify adhesion-related proteins in each matrix. p-LG contained the least adhesion proteins relative to Myogel and h-LG and had only a few proteins in common with these matrices. This might explain why a low number of cells attached to p-LG compared with other matrices. Myogel and h-LG shared 42 proteins, however, 31 adhesion proteins were only found in h-LG, in which we saw a difference between primary tumour and metastatic cell adhesion. Among unique adhesion proteins in h-LG were 12 proteins reported to localize outside the cell, in cell junctions or in extracellular matrix. These proteins included, for example, leucine-rich alpha-2-glycoprotein, annexin A1, annexin A2, galectin-3-binding protein, laminin subunit gamma-2 and plakophilin-2. These proteins have recently been linked to cancer progression. Overexpression of leucine-rich alpha-2-glycoprotein in malignant tumours has been shown to promote angiogenesis and EMT and inhibit apoptosis of cancer cells . The expThis study has potential limitations. Use of several more cell lines would have strengthened our conclusions on the effects of matrix on OTSCC cell adhesion and invasion patterns. However, due to unavailability of resources we were not able to include more than three primary-metastatic cancer cell lines to our experiments. Furthermore, since h-LG was done by combining material from more than one patient the variation in protein expression between patients was lost. In the future, a set of h-LGs produced from one patient could be analysed to find common proteins that might affect OTSCC cell behaviour. The wide range of standard deviations can be explained by high biological variation, which is natural to aggressive cancer cells, especially since the patient-derived cell lines we have used are not fully established. Furthermore, we report the combined averages of three or more experiments, which evidently increases the SD. Finally, the matrices could have been compared to widely used MatriGel, but since we have done this in our previous publications , 17, 18 To increase the reliability of cancer cell experiments in vitro, it is critical to study the cells under biologically relevant conditions, understand the effects of TME and choose the appropriate matrix based on the cell lines and the type of assays. Here, we demonstrated that human HNSCC patients’ lymph node-derived matrix is suitable for studying metastatic oral tongue squamous cell carcinoma cells. It could be used for various 3D assays, e.g. spheroid invasion, in scratch wound migration and in invasion assays. In the future, it should be investigated whether this novel h-LG matrix could be useful in LN metastatic cancer’s drug screening and for personalized cancer medicine in vitro assays.Below is the link to the electronic supplementary material.Additional file 1: Excel TablesAdditional file 2: Supplementary Figures


Veronica species  and to test their antiproliferative and apoptotic activities, according to the authors’ knowledge, because of insufficient research and lack of information. Also, the goal was to determine which obtained samples were better in achieving antiproliferative and apoptotic activities and due to which volatile components. Therefore, essential oils (EOs) and hydrosols (HYs) were isolated from the above-mentioned Veronica species by microwave-assisted extraction (MAE). Phytochemical identification of the free volatile compounds was performed using a GC equipped with a flame ionization detector and a mass spectrometer. Their antiproliferative and apoptotic activities against two human cancer cell lines, breast cancer cell line MDA-MB-231 and bladder cancer cell line T24, were determined. The main compounds identified in the studied Veronica EOs and HYs were terpinen-4-ol (0.34–6.49%), linalool (0.34–6.61%), (E)-caryophyllene (0.97–7.55%), allo-aromadendrene (0.18–2.21%), caryophyllene oxide (1.42–23.83%), benzene acetaldehyde (0.26–13.34%), and β-ionone (1.08–16.53%). In general, HYs of the tested Veronica species showed higher antiproliferative activity (IC50 13.41–42.05%) compared to EOs (IC50 158.1–970.4 µg/mL) on MDA-MB-231 and T24 cancer cell lines after 48 and 72 h. V. agrestis EO showed the best apoptotic effect among the EOs on the MDA-MB-231 cancer cell line  and among the HYs V. cymbalaria showed 9.95 ± 1.05% and 3.06 ± 0.28% of early/late apoptosis and V. anagalloides 8.29 ± 1.09% and 1.95 ± 0.36% of early/late apoptosis compared with control  on the T24 cancer cell line. Future research will include other Croatian species of the genus Veronica to gain a more complete insight into the biological activity of the volatile products of this genus for potential discovery of drugs based on natural plant extracts.This study was conducted to determine the differences in the chemical composition of the essential oils and hydrosols of six different  Veronica, which has recently been studied because of its characteristic specialized metabolites, formerly a member of the Scrophulariaceae, was subsequently transferred to the Plantaginaceae family after phylogenetic and chemotaxonomic studies [Veronica has mainly focused on iridoid glycosides, flavonoids, and saponins [Veronica species are used in traditional medicine worldwide for the treatment of different disorders such as the following: as an antiscorbutic, for wound healing, in respiratory diseases for cough, or as an expectorant [Veronica species has shown that because of their specialized metabolites, they possess antioxidant, antimicrobial, cytotoxic, and antitumor activities [Veronica species against two tumor cell lines, KB and B16 cells. Their results showed that the MeOH extracts possessed anti-inflammatory and cytotoxic activities. EOs have been demonstrated to possess anticancer properties through different mechanisms, including cancer prevention mechanisms, as well as the impact of the established tumor cell itself and the interaction with the microenvironment [Veronica extracts having been used in traditional medicine for cancer treatment, only a few species have been studied for their cytotoxic and anticancer activity [Veronica were selected for this study.Throughout the long development of civilization, people have selected plants as food or/and medicine, primarily based on organoleptic evaluations. Scientific confirmation of the use of medicinal plants began with the development of analytical methods, especially chromatography. Chromatographic techniques enabled the identification and quantification of components of specialized metabolites from plant extracts and stimulated interest in studying the effects of these natural products on long-term health and preventive treatment . Prior t studies . To datesaponins ,13,14, wsaponins ,16,17. Sectorant . Previoutivities ,20,21. Htivities  demonstrironment . Key feaironment . In mediironment . Despiteactivity . Defectsactivity . TherefoVeronica species distributed in Croatia, V. agrestis L., V. anagalloides Guss., V. austriaca ssp. jacquinii L., V. beccabunga L., V. cymbalaria Bodard, and V. officinalis L., and determination of their antiproliferative and apoptotic activities against two human cancer cell lines: breast cancer cell line MDA-MB-231, and bladder cancer cell line T24. Consequently, the aim of this study was the phytochemical identification of FVCs in EOs and HYs isolated by microwave-assisted extraction (MAE) from six different Veronica species collected in 2022 (Extraction of volatiles from the six Croatian  in 2022  was perfE)-caryophyllene, allo-aromadendrene, caryophyllene oxide, hexahydrofarnesyl acetone, phytol, β-ionone, hexadecanoic acid, docosane, tricosane, tetracosane, and octacosane were detected in six studied Veronica EOs ; in V. anagalloides, the dominant compounds are hexahydrofarnesyl acetone (16.17%) and β-ionone (13.13%). In addition, in the composition of V. austriaca ssp. jacquinii EO, hexadecanoic acid (27.66%) and phytol (13.02%) are the predominant compounds. Phytol and hexadecanoic acid are also the predominant constituents in the species V. beccabunga, with 28.08% and 17.06%, respectively. In addition to these two compounds already mentioned, the compound caryophyllene oxide (23.83%) is significantly present in the species V. cymbalaria. Together with phytol and hexadecanoic acid, which are also significantly present in the EO composition of V. officinalis, heptacosane is the most abundant compound (17.21%) (Peculiarities in the EO composition for each species were investigated. In the composition of (17.21%) .4-ol, linalool, (E)-caryophyllene, allo-aromadendrene, caryophyllene oxide, benzaldehyde, benzene acetaldehyde, and β-ionone were detected in the six studied Veronica HYs , (E)-β-damascenone (12.42%), benzene acetaldehyde (11.56%), and β-ionone (10.32%). In V. anagalloides HY, β-ionone (16.53%), benzaldehyde (13.56%), methyl eugenol (13.57%), and (E)-β-damascenone (11.55%) dominate. In addition, methyl eugenol accounts for 37.01%, more than one-third of all identified components in the HY composition of V. austriaca ssp. jacquinii. The HY composition of V. beccabunga differs significantly from the other Veronica species studied, as the HY composition is dominated by α-pinene and piperitone with 17.11% and 19.54%, respectively. The HY of V. cymbalaria is rich in the phenolic constituents methyl eugenol (38.61%) and (Z)-methyl isoeugenol (31.32%). Methyl eugenol also predominates in the HY of V. officinalis with 22.01% and is the most abundant compound, followed by the compounds (E)-β-damascenone and β-ionone with an identification percentage of more than 14%  after MAE for 4, 24, 48, and 72 h. After MTT cell proliferation assay, obtained results were expressed as % of metabolically active cells . In this work, only results for the times at which the samples showed antiproliferative activity in tested concentrations and dilutions are presented. In Cell viability and proliferation after treatment with essential oils (EOs) and hydrosols (HYs) of six ve cells  and IC50V. agrestis EO on MDA-MB-231 after 48 and 72 h was IC50 572.1 µg/mL and 586.9 µg/mL  and IC50 340.6 µg/mL on T24 after 72 h   a,b. On t and 50% c.V. anagalloides EO showed antiproliferative activity after 48 and 72 h on the MDA-MB-231 cancer cell line with IC50 180.1 µg/mL and 243.4 µg/mL  and T24 cancer cell line was IC50 389.9 µg/mL and 158.1 µg/mL   and 33.70%, 26.96%, and 31.76% after 24, 48, and 72 h on T24 cancer cell lines  (0 µg/mL) a,b. HY oand 50%) c,d.V. austriaca ssp. jacquinii EO showed antiproliferative activity only after 72 h on both mentioned cancer cell lines as IC50 816.0 µg/mL on MDA-MB-231  and 617.1 µg/mL on T24  . After 48 h on MDA-MB-231, it showed an antiproliferative effect of 42.05% but pointed to the T24 cell line antiproliferative activity of 33.65%, 26.72%, and 23.58%  (Species 0 µg/mL) a,b. In gand 50%) c,d. V. beccabunga HY showed only antiproliferative effect on T24 cancer cell line, IC50 36.05%, 29.64%, and 39.29% after 24, 48, and 72 h, and metabolically active cells are presented in V. beccabunga did not show any antiproliferative effect.V. cymbalaria showed antiproliferative effect on the MDA-MB-231 cancer cell line (IC50 249.9 µg/mL and 841.3 µg/mL after 48 and 72 h)  compared to V. cymbalaria EO on the T24 cancer cell line (IC50 970.4 µg/mL after 72 h)    (EO of 0 µg/mL) a,b. HY oand 50%) c,d.V. officinalis EO did not show antiproliferative activity at all four tested times , while V. officinalis HY showed excellent antiproliferative activity on MDA-MB-231 and T24 cancer cell lines. V. officinalis HY showed on MDA-MB-231 an antiproliferative effect of the cells after 48 and 72 h   and after 24, 48, and 72 h on the T24 cancer cell line as IC50 21.83%, 13.41%, and 15.22%  (It is interesting that the and 50%) a,b. Veronica species that showed antiproliferative activity after 48 h was determined on two cancer cell lines: breast cancer cell line MDA-MB-231 and bladder cancer cell line T24. Results are expressed as the distinction between early (Annexin-V+/PI−) and late (Annexin-V+/PI+) apoptotic cells. All results are expressed as mean ± SD (n = 3). V. agrestis EO showed apoptotic activity on MDA-MB-231 of 10.47 ± 0.53% and 9.06 ± 0.74%, while V. agrestis HY showed 1.37 ± 0.33% and 0.40 ± 0.09% in early apoptosis and late apoptosis  and hydrosols (HYs) of some poptosis a,b, resppoptosis a,b, respell line . V. cymbectively a. HY of ectively b. V. offpoptosis , respectV. anagalloides EO was 6.1 ± 1.79% and 0.05 ± 0.0.5% . Twelve samples were obtained—two samples for each species  and hydrosol (HY)). All samples were analyzed by gas chromatography–mass spectrometry (GC-MS), and the obtained data are presented in Veronica species was collected in 2022  of six Croatian  in 2022 .V. agrestis were analyzed for the first time, and phytol was the dominant component in the fraction EO with 56.57% (E)-β-damascenone (12.42%), benzene acetaldehyde (11.56%), and β-ionone (10.32%)  . Veronica species studied in this paper were compared with previously published data [V. anagalloides, the dominant compounds are hexahydrofarnesyl acetone (16.17%) and β-ionone (13.13%). Comparing the composition of the oil components of this species with the data previously published in the article by Dunkić et al. [V. anagalloides was 14.33% for HD and 19.12% for MAE [β-ionone relative percentage was found in this sample obtained by MAE compared to this study: only 4.22%.The FVCs of the other five hed data ,7,28. InV. austriaca ssp. jacquinii EO, hexadecanoic acid (27.66%) and phytol (13.02%) were the predominant compounds (V. austriaca ssp. jacquinii in MAE extract (22.17%) [V. beccabunga, with 28.08% and 17.06%, respectively. The dominant compound in V. beccabunga collected in 2021 is oxygenated diterpene phytol with 34.54% for MAE [In the composition of ompounds . Hexadecoic acid 7.66% andoic acid 7.66% andoic acid 7.66% andV. cymbalaria with 3.71% in 2021 and 16.66% in 2022   , while iV. anagalloides species are the following: in the extracts from 2022, the compound β-ionone was 16.53% and (E)-β-damascenone was 11.55% (β-ionone with 6.07% and (E)-β-damascenone with 1.52% [V. anagalloides, V. austriaca ssp. jacquinii, V. cymbalaria, and V. officinalis, could also be related to different locations of material collection, not just the difference in the year of collection.The most significant differences in the proportions of the following compounds in the HY of s 11.55% , while iV. austriaca ssp. jacquinii from 2022, while it was not identified at all in the year before; moreover, not a single phenolic component characteristic of species of the genus Veronica was identified [V. beccabunga HY is dominated by α-pinene and piperitone at 17.11% and 19.54%  and (Z)-methyl isoeugenol (31.32%), while oxygenated sesquiterpene caryophyllene oxide is 6.26% -methyl isoeugenol was identified with less than 1% [V. officinalis with 22.01%, while in the previously published composition, the hydrosols of phenolic compounds was the only one represented by 2-methoxy-4-vinylphenol with 11.12%. The compounds (E)-β-damascenone and β-ionone for V. officinalis were identified in a similar percentage in both years compared . V. agrestis EO showed significant antiproliferative activity on MDA-MB-231 after 48 and 72 h (572.1 µg/mL and 586.9 µg/mL), and 340.6 µg/mL on T24 after 72 h. According to Nazlić et al. [V. saturejoides (Kamešnica Sample) on the HeLa, HCT116, and U2OS cell lines. EO of V. anagalloides showed the highest antiproliferative activity among all tested EOs . Nazlić et al. [V. austriaca ssp. jacquinii EO showed antiproliferative activity above 1000 µg/mL after 48 h on the tested cell lines, while in our present study, the EO of this species showed slightly better results, but after 72 h on both mentioned cancer cell lines (816.0 µg/mL on MDA-MB-231 and 617.1 µg/mL on T24). EO of V. cymbalaria showed an antiproliferative effect on the MDA-MB-231 cancer cell line , while V. cymbalaria EO showed lower activity on the T24 cancer cell line (IC50 970.4 µg/mL after 72 h). According to Nazlić et al. [V. officinalis EO showed significant antiproliferative activity on the HCT116 (IC5O > 500 µg/mL), HeLa, and U2OS cancer cell lines (IC5O > 1000 µg/mL). That could be a reason why the EOs of V. officinalis and V. beccabunga did not show antiproliferative activity in this study, as the intervals of tested concentrations were 50–1000 µg/mL. Generally, V. agrestis EO, V. anagalloides EO, and V. cymbalaria EO showed the highest antiproliferative activity of all tested Veronica species’ EOs. This could be due to the higher relative percentage of phytol, (E)-caryophyllene, and caryophyllene oxide in these species. For these compounds, significant antiproliferative and/or apoptotic activity was reported in recent studies [So far, only a few onducted ,30. In tć et al.  reported MDA-MB-2 and T24. studies ,33.V. anagalloides, V. austriaca ssp. jacquinii, and V. cymbalaria showed antiproliferative activity on MDA-MB-231 only after 48 h (17–43%). V. beccabunga HY did not show an antiproliferative effect at the tested dilutions and times on the MDA-MB-231 cancer cell line. HYs of V. anagalloides, V. austriaca ssp. jacquinii, V. cymbalaria, and V. beccabunga showed high antiproliferative activity (18–40%), especially on the T24 cancer cell line after 24, 48, and 72 h. In this study, HY of V. officinalis was the most effective in antiproliferative activity on the MDA-MB-231  and T24 cell lines . These results are in accordance with previous results on HYs of V. officinalis on HeLa, HCT116, and U2OS [V. agrestis did not show antiproliferative activity on the T24 cancer cell line. The reason for its biological inactivity potentially lies in the smaller amount of plant material from which the HY was obtained. The reason for the excellent antiproliferative activity of V. officinalis HY could be the presence of methyl eugenol, terpinen-4-ol, linalool, and β-ionone in higher relative percentages than in other tested HYs. For these compounds, significant antiproliferative and/or apoptotic activity was reported in recent studies [β-ionone was present in a higher percentage in V. anagalloides and V. beccabunga HYs, which could be the explanation for the great antiproliferative activity shown for these extracts, especially on the T24 cancer cell line. V. beccabunga HY also contained α-pinene in a high percentage, which could be explanation for the great antiproliferative activity on the T24 cancer cell line. There are many more studies of antiproliferative activity on various phenolic extracts of the Veronica species than on EOs and HYs. Harput et al. [V. hederifolia, V. cymbalaria, V. persica, V. pectinata var. glandulosa, and V. polita methanolic extracts showed cytotoxic, anti-inflammatory, and radical-scavenging activities. V. americana methanolic extract showed antiproliferative activity on two cancer cell lines, HF-6 (colon cancer) and PC-3 (prostate cancer) [V. persica Poir, V. anagallis-aquatica, and V. thymoides subsp. pseudocinerea showed cytostatic and cytotoxic activities on human and murine cancer cell lines, with verminoside being the most cytotoxic component. Another study on V. cuneifolia subsp. cuneifolia and V. cymbalaria aqueous extracts showed moderate antiproliferative activity on Hep-2 (human epidermoid carcinoma), RD (human rhabdomyosarcoma), and L-20B (transgenic murine L-cells) [HYs of and U2OS . HY of V studies ,37,38,39ivity 18–%, especiL-cells) . Veronica species that have been tested for apoptotic activity. In general, EOs have been shown to induce intrinsic (mitochondria-dependent) and extrinsic (or cell death receptor-dependent) pathways of apoptosis. A study on the antitumor properties of caffeine isolated from the EO of Piper cernuum in melanoma cells showed that this component could induce apoptosis by activating the caspase-3 pathway, as well as by activating endoplasmic reticulum (ER) stress signaling. Another study focused on evaluating the mechanism of the action of carvacrol found in oregano and thyme EO. Carvacrol induced apoptosis in the MDA-MB-231 breast cancer cell line via mitochondrial membrane permeabilization resulted in the release of cytochrome C, and the induction of caspases was indicated by DNA cleavage and fragmentation [Veronica sibirica (Vtfs) induced dose-dependent apoptosis in MCF-7 breast cancer cells with IC50 of 42 µg/mL. In our present study, apoptotic activity was tested on the previously mentioned Veronica species on MDA-MB-231 and the T24 cancer cell line. Mastelić et al. [V. agrestis EO on the MDA-MB-231 cancer cell line , and among the HYs, V. cymbalaria showed 9.95 ± 1.05% of early apoptotic and 3.06 ± 0.28% of late apoptotic cells, and V. anagalloides 8.29 ± 1.09% of early apoptotic and 1.95 ± 0.36% of late apoptotic cells, comparing to control  on the T24 cancer cell line. To our knowledge, no other study involved testing apoptotic activity of the Veronica species’ EOs or HYs. Induction of apoptosis is extremely desirable for cancer control . As far entation . Accordientation , flavonoć et al.  reportedć et al. , apoptotć et al. , cisplatVeronica species, and significant antiproliferative and apoptotic activities of tested extracts and their possible chemotherapeutic properties, which is why they deserve further investigation. Also, one of the most extensive and interesting fields of application of nanobiotechnology is medicine using plant extracts. Ahmadov et al. [Scutellaria baicalensis extract, and the components present in plant extract were the most important in the formation and stabilization of silver nanoparticles. These bioactive components can be connected to the surface of the silver nanoparticles and function as nanodrugs. Therefore, future research should be focused on FVCs present in the Veronica species on antiproliferative and apoptotic activities, their possible encapsulation for in vivo studies, and as potential nanodrugs in medicine.The results obtained in this research showed comparison of EO and HY composition from the above-mentioned v et al.  reportedVeronica species were collected during the flowering period in May and June 2022 at various locations in Croatia  of each  studied  was hydr−1 and maintained for 25 min . The condition for the CP Wax 52 column was a temperature of 70 °C  for 5 min, which was then increased to 240 °C at a rate of 3 °C min−1 and maintained for 25 min . The injection volume was 2 µL, and the split ratio was 1:20. The MS conditions were as follows: ion source temperature, 200 °C; ionization voltage, 70 eV; mass scan range, 40–350 mass units. The individual peaks of all samples were identified by comparing their retention indices of n-alkanes with those of authentic samples and the studies [Veronica species [n = 3 ± SD).Chromatographic analyses were performed using a GC  equipped with a flame ionization detector and a mass spectrometer , a nonpolar capillary column VF-5 ms , and a polar CP Wax 52 . The chromatographic methods and conditions for hydrosol fraction analysis were the same as described in the article by Dunkić et al. [ studies ,48 by co species ,50,51. RVeronica species  were performed against two human cancer cell lines, breast cancer cell line MDA-MB-231 and bladder cancer cell line T24, to determine which sample has the best antiproliferative activity. Stock solutions of EOs were prepared in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL. Cell lines MDA-MB-231 and T24 were grown in Dulbecco’s modified Eagle’s medium  in a humidified incubator at 37 °C with 5% CO2. The DMEM medium contained 10% fetal bovine serum  and 1% antibiotics  and was used as a negative control. An equal number of cells (1 × 104) were transferred into 96 wells and left overnight. The cells were then treated with EOs of the above-mentioned Veronica species at concentrations of 50, 100, 250, 500, and 1000 µg/mL, while HYs were tested at different dilutions  after MAE for 4, 24, 48, and 72 h. Cell viability and proliferation were determined by measuring cellular metabolism using an MTT assay. Yellow tetrazoline MTT -2,5-diphenyltetrazoline bromide) is reduced in metabolically active cells to the purple formazan. After 2 h, the medium with MTT was removed, and DMSO was added. The plates were incubated for 10 min at 37 °C with shaking. The absorbance was measured at 570 nm by a HiPo MPP-96 microplate photometer . All samples were run on three different plates in triplicate per plate. Solvent control was measured: the highest concentration of DMSO was adjusted to 1% (v/v) and did not show antiproliferative activity. According to Mastelić et al. [t-test with unequal variances was performed using GraphPad Prism 9.0 statistical software  with the significance set at * p < 0.05. The calculation of IC50 values was performed with GraphPad Prism software version 9.0 , normalizing the data by three independent measurements of untreated controls.Cell viability and proliferation after treatment with essential oils (EOs) and hydrosols (HYs) of six different ć et al. , the antć et al. , cisplat4) were seeded in 6-well plates and treated with essential oils (EOs) and hydrosols (HYs) of V. agrestis, V. anagalloides, V. austriaca ssp. jacquinii, V. beccabunga, V. cymbalaria, and V. officinalis for 48 h and then analyzed for apoptosis to determine which sample has the best apoptotic activity. The antiproliferative activities of V. agrestis EO and HY on the MDA-MB-231 cancer cell line after 48 were IC50 572.1 µg/mL and 28.43%. V. anagalloides EO showed antiproliferative activity after 48 on the MDA-MB-231 and T24 cancer cell lines . HY of V. anagalloides showed antiproliferative activity of 19.82% on MDA-MB-231 and of 26.96% after 48 h on T24 cancer cell lines. V. austriaca ssp. jacquinii HY showed better antiproliferative effect, especially on the T24 cancer cell line. After 48 h on MDA-MB-231, it showed antiproliferative effect of 42.05%, but it pointed to the T24 cell line antiproliferative activity of 26.72% after 48 h. V. austriaca ssp. jacquinii HY showed on MDA-MB-231 an antiproliferative effect of 42.05% and pointed to the T24 cell line an antiproliferative activity of 26.72% after 48 h. V. beccabunga HY showed an antiproliferative effect on the T24 cancer cell line of 29.64% after 48 h. EO of V. cymbalaria also showed antiproliferative activity on the MDA-MB-231 cancer cell line (IC50 249.9 µg/mL) after 48 h. HY of V. cymbalaria showed antiproliferative effect on the MDA-MB-231 and T24 cancer cell lines . V. officinalis HY showed on the MDA-MB-231 and T24 cancer cell lines significant antiproliferative effect after 48 h . A combination of Annexin-V-FITC and propidium iodide staining allows the distinction between early (Annexin-V+/PI−) and late (Annexin-V+/PI+) apoptotic cells, necrotic cells, and live cells. After treatment with EOs and HYs, the cells were trypsinized, washed with PBS, and resuspended in 100 µL of the binding buffer containing 5 µL of Annexin-V-FITC and/or 10 µL of PI . The cells were incubated for 15 min at room temperature in the dark and, thereafter, analyzed by flow cytometry . Using the FlowLogic Software , the percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD). For statistical analyses of the apoptosis rate, a t-test with unequal variances, one-way ANOVA followed by a post hoc Tukey test or Kruskal–Wallis test, followed by Dunn’s post hoc test, were performed using GraphPad Prism 7.0 statistical software , with the significance set at p < 0.05, p < 0.01 and p < 0.001.An equal number of cells (1 × 10Veronica species were obtained by microwave-assisted extraction (MAE). The identification of free volatile compounds (FVC) from six Veronica species was analyzed using gas chromatography–mass spectrometry, and the composition of the compounds was compared. The components identified in the EO extracts of all six Veronica species studied are linalool, (E)-caryophyllene, allo-aromadendrene, caryophyllene oxide, phytol, benzene acetaldehyde, β-ionone, hexadecanoic acid, docosane, tricosane, tetracosane, and octocosane. The constituents terpinen-4-ol, (E)-caryophyllene, allo-aromadendrene, caryophyllene oxide, benzaldehyde, benzene acetaldehyde, and β-ionone were identified in all studied HY extracts. These volatile compounds belong to the specialized metabolites, and it is important to know their composition for further research on the pharmaceutical activity of the Veronica species. Also, the antiproliferative and apoptotic effects of essential oils and hydrosols were compared. Generally, hydrosols showed better antiproliferative activity, especially on the T24 cancer cell line. Among the tested EOs, V. anagalloides exerted the best antiproliferative effect on both the MDA-MB-231 and T24 cancer cell lines, while among the tested HYs, V. officinalis HY showed the best antiproliferative activity. The best apoptotic effect of the EOs was shown by V. agrestis EO on the MDA-MB-231 cancer cell line, and among the HYs, V. cymbalaria and V. anagalloides showed the best proapoptotic activities on the T24 cancer cell line. Future research should be focused on the discovery of drugs based on natural plant extracts, which implies testing the combinatorial effect of compounds that show biological activity.Free volatile compounds (FVCs) present in essential oils (EOs) and hydrosols (HYs) of six