Poor prenatal development, followed by rapid childhood growth, conveys greater cardiometabolic risk in later life. Microswine offspring exposed to perinatal maternal protein restriction [MPR; "low protein offspring" (LPO)] grow poorly in late-fetal/neonatal stages. After weaning to an ad libitum (AL) diet, LPO-AL exhibit accelerated growth and fat deposition rates with low adiponectin mRNA, despite low-normal body fat and small intra-abdominal adipocytes. We examined effects of caloric restriction (CR) on growth and metabolic status in LPO and normal protein offspring (NPO) randomized to AL or CR diets from weaning. CR transiently reduced growth in both LPO and NPO, delaying recovery in female LPO-CR. Over 7.5-12.5 weeks, linear growth rates in LPO-CR were slower than LPO-AL ( P < 0.001) but exceeded NPO-AL; body weight growth rates fell but were lower in LPO-CR versus NPO-CR. Linear acceleration ceased after 12 weeks. At 16 weeks, percent catch-up in LPO-CR was reduced versus LPO-AL ( P < 0.001). Plasma growth hormone was low in LPO ( P < 0.02). CR normalized fat deposition rate, yet adiponectin mRNA remained low in LPO-CR ( P < 0.001); plasma adiponectin was low in all LPO-AL and in female LPO-CR. Insulin sensitivity improved during CR. We conclude that in LPO: 1) CR delays onset of, but does not abolish, accelerated linear growth, despite low growth hormone; 2) CR yields stunting via delayed onset, plus a finite window for linear growth acceleration; 3) MPR lowers adiponectin mRNA independently of growth, adiposity, or adipocyte size; and 4) MPR reduces circulating adiponectin in LPO-AL and female LPO-CR, potentially enhancing cardiometabolic risk.

Lactation insufficiency is variously defined and includes the inability to produce milk, not producing enough milk to exclusively meet infant growth requirements, and pathological interruption of lactation (e.g., mastitis). Of women with intent-to-breastfeed, lactation insufficiency has been estimated to affect 38%–44% of newly postpartum women, likely contributing to the nearly 60% of infants that are not breastfed according to the World Health Organization’s guidelines. To date, research and clinical practice aimed at improving feeding outcomes have focused on hospital lactation support and education, with laudable results. However, researchers’ reports of recent rodent studies concerning fundamental lactation biology have suggested that the underlying pathologies of lactation insufficiency may be more nuanced than is currently appreciated. In this article, we identify mucosal biology of the breast and lactation-specific liver biology as two under-researched aspects of lactation physiology. Specifically, we argue that further scientific inquiry into reproductive state-dependent regulation of immunity in the human breast will reveal insights into novel immune based requirements for healthy lactation. Additionally, our synthesis of the literature supports the hypothesis that the liver is an essential player in lactation—highlighting the potential that pathologies of the liver may also be associated with lactation insufficiency. More research into these biologic underpinnings of lactation is anticipated to provide new avenues to understand and treat lactation insufficiency.

Women diagnosed with breast cancer within 10 years of a completed pregnancy are 2~3x more likely to develop liver metastases than never-pregnant (nulliparous) patients, even after controlling for prognostic variables (Goddard 2017). This finding suggests a unique biology in the postpartum liver, a putative pre-metastatic niche, which makes postpartum patients more susceptible to liver metastases. In rodent models, we previously reported increased liver size, hepatocyte proliferation, and anabolic metabolism during pregnancy and lactation. Within one week post-weaning, the rodent liver returned to its pre-pregnant size via a coordinated cell death and tissue remodeling process we call liver involution (Goddard 2017). We find that this process of involution supports overt liver metastasis using two different mammary tumor cell lines. In women, a liver-breast interaction during pregnancy and weaning, as occurs in rodents, has not been described. To investigate the human liver with pregnancy and weaning, we conducted a prospective, non-interventional clinical trial in healthy pregnant women. 47/77 women completed the pregnancy study visits and 17/47 completed post-wean visits. Participants underwent magnetic resonance imaging (MRI) of their livers, provided blood samples, and completed body metric analyses at each study time point. The majority of women (~70%) had increased liver size and evidence of increased liver function at third trimester compared to first trimester, with liver size returning to pre-pregnancy levels with weaning. In this cohort, the increase in liver size exceeded levels expected if the metabolic demands associated with increased body weight during pregnancy were the primary mediator. Intriguingly, primary bile acid and bile acid synthetic enzyme concentrations correlated with liver size increase during pregnancy. Women whose liver growth did not follow the most often observed pattern of increase with pregnancy and decrease postpartum had unchanged bile acid levels, were more likely to have gestational hypertension, and did not have expected increases in liver glucose production during pregnancy. Combined, this study provides the first description of human liver size increase with pregnancy, and return to pre-pregnant baseline size after weaning, with evidence that women who do not experience these liver changes may be metabolically distinct. Furthermore, these data are consistent with the hypothesis that reproductive alterations to the liver, in particular weaning-induced involution, contributes to the increased risk for liver metastases in women diagnosed with breast cancer postpartum. Reference: Goddard, E. T., Hill, R. C., Nemkov, T., D'Alessandro, A., Hansen, K. C., Maller, O., Mongoue-Tchokote, S., Mori, M., Partridge, A. H., Borges, V. F., & Schedin, P. (2017, Feb). The Rodent Liver Undergoes Weaning-Induced Involution and Supports Breast Cancer Metastasis. Cancer Discov, 7(2), 177-187. https://doi.org/10.1158/2159-8290.CD-16-0822 Citation Format: Alexandra Quackenbush, Erica Goddard, Claire Dorfman, Kimberly Vesco, Jonathan Purnell, Pepper Schedin. Evidence for reproductive control of liver size with implications for risk of liver metastases in postpartum breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-39.

BackgroundCancer patients with liver metastases have limited treatment options, especially as only 15–20% are eligible for curative-intent surgical resection.1 Unfortunately, liver metastases also seem to be poorly responsive to immune checkpoint inhibitors (ICI)].2 3 It could be that the unique immunological hallmarks of the liver, including resident macrophages and significant numbers of NK and NKT cells, create a tumor microenvironment that is best suited to alternative forms of immunotherapy that do not rely exclusively on ICI.MethodsWe investigated how the presence of T, natural killer (NK), and NKT cells impact overt liver metastases using a model in which tumor cells are delivered to the liver via intraportal injection to hosts that were either wiltype, nude, or nude with NK-depletion. NK cell depletion was achieved via administration of anti-asialo GM1 antibody 2 days before tumor cell injection and for the duration of the experiment until endpoint at 6 weeks post tumor cell injection, with NK cell depletion confirmed by flow cytometry. Tumors were assessed histologically.ResultsUsing the portal vein model in female nulliparous mice, overt liver metastasis incidence was about 30% across 2 different mammary tumor cell lines. The incidence rose to 80–100% when tumor cells were delivered to hosts in the post-wean window (referred to as involution hosts), mirroring increased breast cancer metastasis to the liver observed in postpartum breast cancer patients.4 Conversely, when tumor cells were delivered to nude hosts, either nulliparous or involution stages, the incidence of metastases dropped to 0–10%. Importantly, tumor cells injected into the mammary gland of nude mice grew robustly with 100% take. Nude hosts lack T cells and NKT cells; however, NK cells are present. Furthermore, the liver is enriched for NK cells, whilst the mammary gland has few NK cells.5 We hypothesized that NK cells, when in the background of T- and NKT-cell depletion (i.e. nude host), restrict outgrowth of mammary tumor cells in the liver. Six weeks after portal vein injection of mammary tumor cells to nude hosts we find increased incidence of metastasis in the NK-depleted group compared to isotype control, as well as increased number of metastases per mouse.ConclusionsOur data suggest that NK cells play an important role in controlling liver metastases in nude hosts, and that NK activity in wild type hosts is insufficient to control liver metastases. Increasing NK cell cytotoxic activity could be an effective immunotherapy strategy to control liver metastases.ReferencesNordlinger B, Sorbye H, Glimelius B, Poston GJ, Schlag PM, Rougier P, Bechstein WO, Primrose JN, Walpole ET, Finch-Jones M, et al: Perioperative FOLFOX4 chemotherapy and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial. Lancet Oncol 2013;14(12):1208–1215.Bilen MA, Shabto JM, Martini DJ, Liu Y, Lewis C, Collins H, Akce M, Kissick H, Carthon BC, Shaib WL, et al: Sites of metastasis and association with clinical outcome in advanced stage cancer patients treated with immunotherapy. BMC Cancer 2019;19(1):857.Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Sosman JA, Atkins MB, Leming PD, et al: Five-year survival and correlates among patients with advanced melanoma, renal cell carcinoma, or non-small cell lung cancer treated with nivolumab. JAMA Oncol 2019.Goddard ET, Hill RC, Nemkov T, D’Alessandro A, Hansen KC, Maller O, Mongoue-Tchokote S, Mori M, Partridge AH, Borges VF, et al: The rodent liver undergoes weaning-induced involution and supports breast cancer metastasis. Cancer Discov 2017;7(2):177–187.Shi FD, Ljunggren HG, La Cava A, Van Kaer L. Organ-specific features of natural killer cells. Nat Rev Immunol 2011;11(10):658–671.

A postpartum diagnosis of breast cancer in women ≤45 yoa is an independent predictor of liver metastasis, suggesting a liver metastatic advantage (PMID: 27974414). In rodents, we identified a postpartum liver biology that supports liver metastasis in mouse models. This biology is weaning-induced liver involution and is characterized by a transient period of hepatocyte cell death, catabolic metabolism, immune influx, and stromal remodeling (PMID: 27974414). Here, we sought to investigate the mechanism by which post-wean liver involution supports breast cancer metastasis. Liver metastases were induced by portal vein injection of mouse mammary tumor cells into nulliparous or post-wean, involution hosts (PMID: 28060292). Compared to nulliparous mice, involution mice had increased incidence of liver metastasis, as well as more liver lesions per mouse. However, there was no group differences in tumor size or proliferation index. These data are consistent with the involution liver providing an environment that promotes tumor cell establishment, not proliferation. To further investigate, we intraportally injected GFP-tagged tumor cells into GFP-tolerant mice, and assessed tumor cell abundance in the liver at 90 minutes, and 1, 3, and 14 days post injection. Contrary to our expectations, the nulliparous group had more tumor cells present at 90 minutes, 1 day, and 3 days post tumor cell injection. We saw the metastatic advantage in the involution group emerge by 14 days after tumor cell injection. We also observed increased abundance of PD1+ CD4 T cells in addition to heightened IL-10 signaling in the involution group at baseline (i.e. no tumor). Based on these observations, we hypothesized that the involution metastatic niche may be characterized by immune suppression, permitting tumor cell escape from immune surveillance. To address if the involution host liver is immune suppressed, we used an in vivo T cell activation assay where we adoptively transferred antigen-specific T cells systemically, and subsequently injected the cognate antigen into the liver of tumor free mice. Five days later, we assessed antigen-specific T cell number as a measure of T cell priming and activation. We found significantly impaired naïve T cell priming in the normal involution host liver compared to the nulliparous host. These data raise the possibility that impaired adaptive immunity could be responsible for increased liver metastasis in the involution group. In support, when we depleted CD8 T cells in our liver metastasis model, we found that metastasis in the anti-CD8 treated nulliparous group was similar to the untreated involution group, showing that depletion of cytotoxic immune cells in nulliparous hosts can recapitulate the involution host metastatic advantage. Taken together, our findings suggest the normal postpartum liver is in an immune suppressed state, which can provide a pro-metastatic advantage to circulating breast cancer cells. Future work will investigate therapeutic targeting of the postpartum liver niche. Citation Format: Alexandra Quackenbush, Nathan Penncok, Pepper Schedin. Immune suppression established by postpartum liver involution promotes liver metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT004.