Author response: Persistent effects of pair bonding in lung cancer cell growth in monogamous Peromyscus californicusArticle Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Epidemiological evidence suggests that social interactions and especially bonding between couples influence tumorigenesis, yet whether this is due to lifestyle changes, homogamy (likelihood of individuals to marry people of similar health), or directly associated with host-induced effects in tumors remains debatable. In the present study, we explored if tumorigenesis is associated with the bonding experience in monogamous rodents at which disruption of pair bonds is linked to anxiety and stress. Comparison of lung cancer cell spheroids that formed in the presence of sera from bonded and bond-disrupted deer mice showed that in monogamous Peromyscus polionotus and Peromyscus californicus, but not in polygamous Peromyscus maniculatus, the disruption of pair bonds altered the size and morphology of spheroids in a manner that is consistent with the acquisition of increased oncogenic potential. In vivo, consecutive transplantation of human lung cancer cells between P. californicus, differing in bonding experiences (n = 9 for bonded and n = 7 for bond-disrupted), and nude mice showed that bonding suppressed tumorigenicity in nude mice (p<0.05), suggesting that the protective effects of pair bonds persisted even after bonding ceased. Unsupervised hierarchical clustering indicated that the transcriptomes of lung cancer cells clustered according to the serum donors’ bonding history while differential gene expression analysis pointed to changes in cell adhesion and migration. The results highlight the pro-oncogenic effects of pair-bond disruption, point to the acquisition of expression signatures in cancer cells that are relevant to the bonding experiences of serum donors, and question the ability of conventional mouse models to capture the whole spectrum of the impact of the host in tumorigenesis. eLife digest People’s social interactions could influence their risk of developing various diseases, including cancer, according to population-level studies. In particular, studies have identified a so-called widowhood effect where a person’s risk of disease increases following the loss of a spouse. However, the cause of the widowhood effect remains debatable, as it can be difficult to separate the impact of lifestyle changes from biological changes in the individual following bereavement. It is not possible to use laboratory mice to identify a causal biological mechanism, because they do not form long-term relationships with a single partner (pair bonds). However, several species of deer mouse form pair bonds, and suffer from anxiety and stress if these bonds are broken. Naderi et al. used these mice to study the widowhood effect on the risk of developing cancer. First, Naderi et al. grew human lung cancer cells in blood serum taken from mice that were either in a pair bond or had been separated from their partner. The cancer cells grown in the blood of mice with disrupted pair bonds changed size and shape, indicating that these mice were more likely to develop cancer. This effect was not observed when the cells were grown in the blood of bonded deer mice or of another deer mouse species that does not form pair bonds. Naderi et al. also found that the activity of genes involved in the cancer cells’ ability to spread and to stick together was different in pair-bonded mice and in pair-separated mice. Next, Naderi et al. implanted lung cancer cells into the deer mice to study their effects on live animals. When cancer cells from the deer mice were transplanted into laboratory mice with a weakened immune system, the cells taken from pair-bonded deer mice were less likely to grow than the cells from deer mice with disrupted pair bonds. This suggests that the protective effects of pair bonding persist even after removal from the original mouse. These results provide evidence for a biological mechanism of the widowhood effect, where social experiences can alter gene activity relating to cancer growth. In the future, it will be important to determine whether the same applies to humans, and to find out if there are ways to mimic the effects of long-term bonds to improve cancer prognoses. Introduction While the psychosomatic impact of cancer in patients is extensively documented, the reciprocal effects of individuals’ social experiences in carcinogenesis receive limited attention. Both anecdotal and experiential evidence, and numerous epidemiological studies, strongly suggest that emotional factors can affect the development and progression of cancer, pointing to the sensitivity of cancer cells to signals associated with behavior, emotional state, and sociality. For example, the marital status modulates the likelihood for the development of fatal cancers, with unmarried, divorced, or widowed individuals exhibiting an increased chance of developing life-threatening disease and males being more susceptible than females to the protective effects of marriage (Aizer et al., 2013). The ‘widowhood effect’ provides an example at which in couples, after the loss of one partner, the surviving one exhibits an increased probability for the development of various fatal pathologies (Elwert and Christakis, 2008; Blanner et al., 2020; Sullivan and Fenelon, 2014; Bowling, 1987; Boyle et al., 2011). Notwithstanding that high variation in death causes has been documented, cancer is recognized as a common cause of mortality (Aizer et al., 2013; Elwert and Christakis, 2008; Blanner et al., 2020; Burgoa, 1998; Martikainen and Valkonen, 1996; Sex, 1973). Although both sexes are influenced by widowhood, males appear more sensitive than females to widowhood-associated death (Sullivan and Fenelon, 2014; Helsing et al., 1981). Despite the information they provide, unavoidable changes in lifestyle habits in the bereaved partner at widowhood or between single and married patients complicate the epidemiological data interpretation. Several mechanisms connecting cancer to social interactions, mental state, and bereavement have been proposed. Laboratory mice of the genus Mus, despite their power in illuminating various aspects of tumorigenesis, remain of limited value in modeling the effects of pair bonding. It is estimated that in less than 10% of mammals, including humans, individuals form pair bonds that are based on mating (Kleiman, 1977; Lukas and Clutton-Brock, 2013; Scribner et al., 2020). Therefore, mice, by not developing long-term pair bonds, are not adequate in studying the effects of widowhood and pair-bond disruption (Chatzistamou et al., 2018; McDonald et al., 2005). Earlier studies in mice have shown that brain-derived signals linked to the reward system may impact tumorigenesis, whereas stress can stimulate metastases (Ben-Shaanan et al., 2018; Sloan et al., 2010). However, more complex behavioral traits involving social interactions in married couples or widowhood cannot be studied in mice. Peromyscus californicus is a monogamous species developing long-term, cohesive pair bonds that can influence various physiological responses (Havighorst et al., 2017; Perea-Rodriguez et al., 2015; Glasper and Devries, 2005; Wright et al., 2018). Upon cyclosporine-mediated immunosuppression, similarly with other rodents, P. californicus supports the growth of human cancers, providing a potentially informative animal model for the study of pair-bond disruption in tumorigenesis in vivo (Fingert et al., 1984; Kaza et al., 2018; Chatzistamou and Kiaris, 2016). Results Bonding history modulates the effects of sera in tumor spheroid formation Initially, we asked if sera of P. californicus following the disruption of pair bonds affected the growth of cancer cells in vitro in a manner that depended on bonding history. We focused on the formation of tumor spheroids that are enriched in cells with cancer stem cell (CSC)-like properties, and their formation is known to reflect tumorigenic activity directly (Visvader and Lindeman, 2012; Ishiguro et al., 2017). Sera were obtained from 14 to 17 months old virgin, bonded for about 12 months, or bond-disrupted (after 12 months of bonding) at the periods indicated, male P. californicus, and the efficacy of spheroid formation by A549 human lung cancer cells was assessed. A pilot study indicated that sera obtained from animals 1 week after the disruption of pair bonds resulted in the formation of larger yet less compact spheroids, suggesting a significant impact of bond disruption in spheroid morphogenesis (Figure 1a). The results were confirmed and extended in a subsequent study that also included sera obtained 24 hr and 2 weeks after the disruption of pair bonds (Figure 1b). In this study, sera from 9 (B), 5 (BD, 24 hr), 5 (BD, 1 week), 4 (BD, 2 weeks), and 5 (virgin, V) different animals were used, and microsphere formation was evaluated in two biological replicas for each (n = 10 for BD [1 week], BD [24 hr], and V; n = 8 for BD [2 weeks] and n = 18 for B). For control media (CM) and plain serum-free media (PM), n = 4. As shown in Figure 1b, this activity was only marginal at 24 hr but was significant (p<0.05) 1 week and 2 weeks after the disruption of pair bonds, implying that the factors responsible accumulated in the sera after pair-bond disruption. As compared to virgins, sera from animals at bonding resulted in the formation of smaller spheroids, albeit insignificantly, which implies that bonding may also have some protective activity, beyond the pro-oncogenic activity of bond disruption (Figure 1b). Figure 1 with 1 supplement see all Download asset Open asset Effects of pair bonding in the pro-oncogenic activity of sera. (a) Representative microphotographs of tumor spheroids developed by A549 cells 3 days after cell seeding. Cells formed spheroids in the presence of sera from bonded (B), bond-disrupted animals (BD) 1 week after disruption, and virgin animals (V), and control media containing fetal bovine serum (FBS) (CM). Live (green) and dead cells (red) are indicated. Representative images of two independent experiments are shown. (b) Representative microphotographs of tumor spheroids developed by A549 cells on day 1, day 2, and day 3, after cell seeding. Cells formed spheroids in the presence of sera from bonded (B), bond-disrupted animals (BD) 24 hr, 1 week, and 2 weeks after disruption, and virgin animals (V), control media containing FBS (CM) or serum-free plain media (PM). The last column shows images at day three in higher magnification. Bars indicate 200 μM. (c) Scatter dot plots of data shown in (b), indicating the size of tumor spheroids at days 2 and 3 after seeding. Median and p-values are indicated. Sera from 9 (B), 5 (BD, 24 hr), 5 (BD, 1 week), 4 (BD, 2 weeks), and 5 (V) different animals were used and microsphere formation was evaluated in two biological replicas for each (n = 10 for BD [1 week], BD [24 hr] and V; n = 8 for BD [2 weeks] and n = 18 for B). For control media (CM) and plain serum-free media (PM), n = 4. For this experiment, sera from 14 to 17 months old mice that were used for the B and BD groups were bonded for about 1 year. Statistical analyses were performed by ANOVA. Variation in spheroid size with sera obtained after bond disruption is due to the genetic diversity of donor animals and persists in different lung cancer cell lines The effects in spheroid size described above were obtained with sera from older animals (14–17 months old) that were bonded for at least 12 months. To test whether disruption of bonds in younger animals that were bonded for shorter time periods also produced similar effects, we conducted the following study: We exposed to sera of 8–10 months old animals that were either pair-bonded for 2 months or following 2 weeks of bond disruption after 2 months of bonding, a roster of lung cancer cell lines. For this experiment, 14 animals were used that represented seven sibling pairs with each sibling allocated either to the bonded or to the bond-disrupted group. Our results indicated that consistently, in the same sibling pair, an induction of microsphere size of similar magnitude was noted for all five cell lines tested, in four of seven pairs, while this effect was only marginal in the remaining three pairs (Figure 1—figure supplement 1). The variation in spheroid size was analogous to that recorded in the results described in Figure 1c. Thus, we conclude that even shorter periods of bonding are sufficient, and the consequences of its disruption can be recorded in sera from even younger animals. More importantly, it indicates that the variation of the effects is due to the diversity of the animals and not to the differential sensitivity of the cell lines used. Persistent pro-oncogenic activity of bond disruption in vivo The effects of pair bonding in spheroid formation prompted us to explore whether bond disruption also influences the efficacy of tumorigenesis in vivo. To that end, vasectomized male P. californicus were allowed to establish pair bonds for about 2 months with their female partners and then subjected to pair-bond disruption (n = 9) or were left with their partners (n = 11). Following immunosuppression by CsA animals were inoculated with A549 human lung cancer cells and tumorigenesis was monitored. Animals that did not possess bonding experiences before were used as controls (n = 8). Tumors grew originally in animals of all experimental groups and by day 15 measurable tumors were detected in 9 of 11 bonded, in 8 of 9 bond-disrupted and in 6 of 8 virgins (Figure 2a). At this point, tumors were modestly – albeit not statistically significantly – larger in the bond-disrupted animals and smaller in the group of virgins (Figure 2a). By day 25, the tumors persisted in both the bond-disrupted and bonded animals, at 89% (8 of 9) and 82% (9 of 11) rate, respectively, while in virgin animals, they were detectable only in 25% (2 of 8) of the animals (Figure 2b,c). Figure 2 Download asset Open asset Growth of A549 human lung cancers in P. californicus (IS stock) and bonding experience. Vasectomized males were used in all studies. (a) Volume of measurable tumors at day 15 following cancer cell inoculation in the bonded (n = 9), bond-disrupted (n = 8), and virgin (n = 6) groups; out of the 11, 9 and 8 animals implanted originally with A549 cells. (b) Pie graphs indicating the percentage of animals bearing tumors at day 25. (c) Representative hematoxylin and eosin (H and E)-stained sections of P. californicus-grown tumors from bonded (upper panel), and bond-disrupted (lower panel) groups. (*) and (#) indicate necrotic areas and muscle invasion, respectively. (d) Tumor-free nude mice implanted with A549 tumor explants from bonded (n = 9) and bond-disrupted (n = 7) P. californicus. p-values (log-rank [Mantel–Cox] test) are shown. (e) H and E-stained sections of A549 tumors in nude mice derived from explants of A549 tumors from bonded, and bond-disrupted P. californicus. The morphology of A549 tumors from the direct inoculation of A549 cells in nude mice is shown (n). N is indicated in the text. B-IS, tumors growing in bonded P. californicus; BD-IS, tumors growing in bond-disrupted P. californicus; B-n, tumors that originally developed in bonded P. californicus and now growing in nude mice; BD-n, tumors that originally developed in bond-disrupted P. californicus and now growing in nude mice; n, tumors that developed in nude mice following injection of A549 cells. In a follow-up study, we explored if differential pro-oncogenic activity persisted after growth in nude mice. Thus, tumors that were originally grown in P. californicus for at least 1 month (n = 9 for bonded and n = 7 for bond-disrupted) were re-transplanted in virgin nude mice (one nude mice for each original Peromyscus tumor), and tumorigenesis was recorded. As shown in Figure 2d, tumors from bonded P. californicus exhibited significantly (p=0.011) lower tumorigenicity in nude mice than those grown originally in the bond-disrupted animals, despite that histologically they remained indistinguishable (Figure 2e). In line with the tumor spheroid analyses, pair bonding produced persistent changes in tumors that suppressed their growth and endured even when bonding seized. Effects of pair bonding in differential gene expression The effects of bonding history in the profile of tumor growth in vivo, combined with the spheroid formation in vitro, imply the induction of transcriptional changes in the cancer cells in a manner that depends on bonding experience (Figure 3, Figure 3—figure supplements 1, 2 and 3). Initially, we focused on the expression of established CSC markers and genes regulating CSC potential, such as Oct-4, b-catenin, and CD-133 that have been identified previously in A549 cells (Chiou et al., 2010; Akunuru et al., 2012; Teng et al., 2010). The analysis was performed by semiquantitative RT-PCR in 2D cultures to eliminate the effects of the clonal selection of cells in the spheroids. Differential expression analysis did not reveal considerable differences between the bonding groups, either in cells cultured in vitro with sera from animals differing in bonding history or in vivo in tumors in nude mice or Peromyscus (Figure 3—figure supplement 1). However, unsupervised hierarchical clustering indicated that these CSC markers provided a signature that predicted a relatively high accuracy bonding history of the animals (Figure 3—figure supplement 1). Figure 3 with 3 supplements see all Download asset Open asset RNAseq analysis of A549 cells cultured in the presence of sera from bonded (B), bond-disrupted (BD), or virgin (V) P. californicus. (a) Bar graphs showing number of differentially expressed genes in each pairwise comparison group. (b ) Volcano plots showing differentially expressed genes between the B vs BD, and B vs V groups. (c) Venn Diagrams showing overlapping differentially expressed genes. The identity of genes is shown in the right. In the bonded group, mice were paired for 12 months. For the bond-disrupted group, we separated paired mice after 12 months of bonding, and collected the sera 1 week after bond disruption. For virgin mice, we collected sera from mice housed 3/cage. This observation prompted us to perform RNA sequencing and analyze expression profiles at the whole transcriptome level in human A549 lung cancer cells in the presence of sera that had been isolated from monogamous male P. californicus that were virgin (V), bonded (B), or subjected to disruption of pair bonds (BD) after bonding (n = 6 samples/group). Controls (C) cultured in the presence of fetal bovine serum (FBS) were also included. Unsupervised hierarchical clustering (Vidman et al., 2019) indicated that the transcriptomes clustered well together according to the serum donors’ bonding history, except the virgin (V) group that exhibited the lowest discrimination (Figure 3—figure supplement 2). Differential gene expression analysis was performed as described before by using the iDEP platform (Ge et al., 2018). This analysis showed that the majority of differentially expressed genes were detected in the comparisons involving the FBS-treated cells (C), which suggests that the species origin of sera produces the most potent effects in gene expression and potentially masking the consequences of pair bonding in the regulation of the transcriptome (Figure 3—figure supplement 3). Thus, we repeated the analysis by excluding the specimens corresponding to FBS and restricted it only to the specimens that received Peromyscus sera (Figure 3). Seven genes were differentially expressed in each B vs BD and B vs V comparisons, while none were detected between the V and BD groups (Table 1). Thus, it seems that pair bonding produces more robust effects in the sera as compared to those of bond disruption. Among these genes, all of which were downregulated in the B group, five were common and included HES1, ZFP36, NR4A1, FGG, and SOCS3. Hes1 is a transcription factor that is downstream of Notch signaling, for which the pro-oncogenic activity in lung cancer has been established (Westhoff et al., 2009; Yuan et al., 2015). NR4A1 encodes for the orphan nuclear receptor for which a with in lung cancer has been shown and is involved in cancer cell et al., 2017; et al., 2018). is a of and is a of lung tumorigenesis et al., and encodes for that has been linked to of lung and other cancer cells et al., 2008; et al., The genes that were detected in the BD vs B groups comparison were and which for A and B and while in the V vs B the that lung cancer cell et al., and the growth factor that at least in lung cancer, is associated with et al., et al., analysis indicated that associated with differentially expressed genes were linked to the regulation of cell and or morphogenesis (Table 2). 1 expressed genes between the BD, and V groups. and are indicated. that are common in the B vs BD and V vs B comparisons are vs B vs B 2 associated with the differentially expressed genes in B vs BD and V vs B groups et al., The p-values are indicated. vs regulation of cell vs regulation of of regulation of regulation of cell of cell and not polygamous Peromyscus are sensitive to the effects of bond disruption in spheroid formation The on P. californicus prompted us to explore whether other Peromyscus species are also sensitive to the effects of the disruption of pair bonds. Thus, we compared the effects of sera from bonded or bond-disrupted polygamous P. and monogamous Peromyscus in the size and of A549 tumor spheroids. As shown in Figure the disruption of pair bonds altered spheroid morphology in the but not in the polygamous The of this effect was the animals and was recorded in at least 6 of 12 male P. polionotus but none of P. (n = Figure supplement 1). to P. californicus at which pair-bond disruption spheroid in P. polionotus the effect was in the that formed in the presence of P. polionotus sera obtained after the disruption of pair bonds had as to the spheroids from P. sera at bonding and bond disruption and those of P. polionotus at bonding that were In some 25% of this was also noted in P. polionotus sera obtained at bonding (Figure supplement 1). this the between the two species or is due to changes in the of the cells and donor animals remains to be In it may reflect the same effect by but recorded at different the formation of the spheroids. It is also that the monogamous in Peromyscus has developed the of P. polionotus and P. californicus, and may have been in the consequences of bond disruption in spheroid formation et al., 2013). To that end, the spheroid size and may be for the two the effects of pair-bond disruption Figure 4 with 1 supplement see all Download asset Open asset spheroids with sera from P. polionotus and P. of tumor spheroids formed after 2, 3, and 7 days in with sera isolated from monogamous P. polionotus and polygamous P. In 6 of 12 P. polionotus but none of 12 P. was noted at bond disruption. For the experiment, sera were obtained from the same animal at bonding for 12 months and after 1 week following bond disruption. animals were housed after from P. BD, P. P. polionotus BD, P. polionotus bond Discussion The present the of the – in its – in cancer progression and the of psychosomatic factors as of cancer growth. a relevant animal results highlight the biological of the ‘widowhood and suggest that it as a beyond lifestyle Our are based on the recorded effects of pair bonding in three of the cancer cells. included tumor spheroid formation established in the presence of sera from bond-disrupted animals, the expression profile of the cancer cells in vitro and in vivo that depended on the bonding history of serum and tumor respectively, and their tumorigenicity in the nude mice. The use of sera from rodents, allowed us to evidence that this effect individuals but persists different lung cancer cells. This observation be of to the study of human that are and their responses to the same may be In animal cancer cells were implanted in animals and the of tumorigenesis was affected by the bonding history. pair bonding and disruption can also influence tumor will have to be the that most cancers are in patients is consistent with the effects of widowhood in the as to the of the by using the in vivo experiments animals mice and study from the of of immune responses that may be especially relevant to widowhood-associated stress. was the loss of the tumors in the virgin animals as to the majority of the bonded and bond-disrupted that (Figure A possible is to the differential of immunosuppression by the after cancer cell may have more immunosuppression in the animals that had been subjected to bonding, due to the of a with in the of social interactions and pair bonding et al., 2005; et al., et al., 2009; Yuan et al., and 2018). It is noted that the high in the tumorigenicity between virgins and the bonded or bond-disrupted animals, differential immune as the for this Differential analysis of gene expression showed that sera from animals at bonding enriched for genes regulating cell and and that are consistent with the recorded changes in spheroid Although for several of the differentially expressed genes, their which was in the bonded group, was associated with a in some it was For example, was downregulated in the bonding group, yet it is a tumor for lung and other cancers et al., and which may reflect responses to bonding et al., its effects in the expression of individual genes, the impact of bonding history in transcription was more in the recorded in the profiles of cells cultured in sera from animals with similar bonding This was especially to the bonded and bond-disrupted groups. is that this is for the lowest in the profile by the serum of virgin animals, as to the changes by the sera of bonded and of bond-disrupted animals that remained more the results provide a for the widowhood effect and suggest that the individuals