Pregnancy and weaning regulate human maternal liver size and function.

During pregnancy, the rodent liver undergoes hepatocyte proliferation and increases in size, followed by weaning-induced involution via hepatocyte cell death and stromal remodeling, creating a prometastatic niche. These data suggest a mechanism for increased liver metastasis in breast cancer patients with recent childbirth. It is unknown whether the human liver changes in size and function during pregnancy and weaning. In this study, abdominal imaging was obtained in healthy women at early and late pregnancy and postwean. During pregnancy time points, glucose production and utilization and circulating bile acids were measured. Independently of weight gain, most women's livers increased in size with pregnancy, then returned to baseline postwean. Putative roles for bile acids in liver growth and regression were observed. Together, the data support the hypothesis that the human liver is regulated by reproductive state with growth during pregnancy and volume loss postwean. These findings have implications for sex-specific liver diseases and for breast cancer outcomes.

Immune evasion of dormant disseminated tumor cells is due to their scarcity and can be overcome by T cell immunotherapies.

The period between "successful" treatment of localized breast cancer and the onset of distant metastasis can last many years, representing an unexploited window to eradicate disseminated disease and prevent metastases. We find that the source of recurrence-disseminated tumor cells (DTCs) -evade endogenous immunity directed against tumor neoantigens. Although DTCs downregulate major histocompatibility complex I, this does not preclude recognition by conventional T cells. Instead, the scarcity of interactions between two relatively rare populations-DTCs and endogenous antigen-specific T cells-underlies DTC persistence. This scarcity is overcome by any one of three immunotherapies that increase the number of tumor-specific T cells: T cell-based vaccination, or adoptive transfer of T cell receptor or chimeric antigen receptor T cells. Each approach achieves robust DTC elimination, motivating discovery of MHC-restricted and -unrestricted DTC antigens that can be targeted with T cell-based immunotherapies to eliminate the reservoir of metastasis-initiating cells in patients.

Current challenges in metastasis research and future innovation for clinical translation.

While immense strides have been made in understanding tumor biology and in developing effective treatments that have substantially improved the prognosis of cancer patients, metastasis remains the major cause of cancer-related death. Improvements in the detection and treatment of primary tumors are contributing to a growing, detailed understanding of the dynamics of metastatic progression. Yet challenges remain in detecting metastatic dissemination prior to the establishment of overt metastases and in predicting which patients are at the highest risk of developing metastatic disease. Further improvements in understanding the mechanisms governing metastasis have great potential to inform the adaptation of existing therapies and the development of novel approaches to more effectively control metastatic disease. This article presents a forward-looking perspective on the challenges that remain in the treatment of metastasis, and the exciting emerging approaches that promise to transform the treatment of metastasis in cancer patients.

Defining cellular population dynamics at single-cell resolution during prostate cancer progression.

Advanced prostate malignancies are a leading cause of cancer-related deaths in men, in large part due to our incomplete understanding of cellular drivers of disease progression. We investigate prostate cancer cell dynamics at single-cell resolution from disease onset to the development of androgen independence in an in vivo murine model. We observe an expansion of a castration-resistant intermediate luminal cell type that correlates with treatment resistance and poor prognosis in human patients. Moreover, transformed epithelial cells and associated fibroblasts create a microenvironment conducive to pro-tumorigenic immune infiltration, which is partially androgen responsive. Androgen-independent prostate cancer leads to significant diversification of intermediate luminal cell populations characterized by a range of androgen signaling activity, which is inversely correlated with proliferation and mRNA translation. Accordingly, distinct epithelial populations are exquisitely sensitive to translation inhibition, which leads to epithelial cell death, loss of pro-tumorigenic signaling, and decreased tumor heterogeneity. Our findings reveal a complex tumor environment largely dominated by castration-resistant luminal cells and immunosuppressive infiltrates.

Association Between Postpartum Breast Cancer Diagnosis and Metastasis and the Clinical Features Underlying Risk.

Importance: In women 45 years or younger, breast cancer diagnosis after childbirth increases the risk for metastasis and death, yet limited data exist to define this window of risk and associated prognostic factors. Objective: To assess the window of elevated risk for metastasis following a postpartum breast cancer (PPBC) diagnosis and whether clinical prognostic factors are associated with the increased risk. Design, Setting, and Participants: This multicenter cohort study conducted using cases from the Colorado Young Women's Breast Cancer Cohort diagnosed between January 1, 1981, and December 31, 2014, included 701 women 45 years or younger with stage I to III invasive breast cancer for whom parity data, including time of last childbirth, were available. Data analysis was conducted from July 1 to September 30, 2017. This study involved a tertiary care academic hospital-based breast center and its regional affiliates with cases from the greater Rocky Mountain region. Exposures: Primary exposures were prior childbirth or no childbirth, time between most recent childbirth and breast cancer diagnosis, and time between breast cancer diagnosis and metastasis. Main Outcomes and Measures: The primary outcome was distant metastasis-free survival. Results: A total of 701 women 45 years or younger from the greater Rocky Mountain states region were included in the analysis; mean (SD) age at diagnosis was 37.9 (5.1) years. Breast cancer diagnosis within 10 years after parturition was associated with elevated risk for metastasis, particularly in women with stage I or II disease. In addition, women with PPBC diagnosed within 10 years of a completed pregnancy that was estrogen receptor-positive showed distant metastasis-free survival similar to that of nulliparous patients with estrogen receptor-negative cancer, and women with estrogen receptor-negative PPBC had further reduced metastasis-free survival. Moreover, women with PPBC had increased lymphovascular invasion and lymph node involvement. In addition, tumor-associated Ki67 positivity identified 129 patients with luminal B cancer in the cohort that, independent of parity status, had poorer prognosis compared with patients with luminal A cancer, although it did not reach statistical significance. Conclusions and Relevance: Diagnosis of PPBC within 10 years post partum appears to be associated with an increased risk for metastasis. This increased risk was highest in stages I and II cancer at diagnosis and present in both patients with estrogen receptor-positive and estrogen receptor-negative cancer, persisting in estrogen receptor-positive cases for up to 15 years after diagnosis. Postpartum breast cancer diagnoses were not associated with increased Ki67 index but were associated with increased lymphovascular invasion and lymph node involvement compared with breast cancer in nulliparous patients.

Targeting the perivascular niche sensitizes disseminated tumour cells to chemotherapy.

The presence of disseminated tumour cells (DTCs) in bone marrow is predictive of poor metastasis-free survival of patients with breast cancer with localized disease. DTCs persist in distant tissues despite systemic administration of adjuvant chemotherapy. Many assume that this is because the majority of DTCs are quiescent. Here, we challenge this notion and provide evidence that the microenvironment of DTCs protects them from chemotherapy, independent of cell cycle status. We show that chemoresistant DTCs occupy the perivascular niche (PVN) of distant tissues, where they are protected from therapy by vascular endothelium. Inhibiting integrin-mediated interactions between DTCs and the PVN, driven partly by endothelial-derived von Willebrand factor and vascular cell adhesion molecule 1, sensitizes DTCs to chemotherapy. Importantly, chemosensitization is achieved without inducing DTC proliferation or exacerbating chemotherapy-associated toxicities, and ultimately results in prevention of bone metastasis. This suggests that prefacing adjuvant therapy with integrin inhibitors is a viable clinical strategy to eradicate DTCs and prevent metastasis.

Dormant tumour cells, their niches and the influence of immunity.

Despite increased focus on the clinical relevance of dormant metastatic disease, our understanding of dormant niches, mechanisms underlying emergence from dormancy, and the immune system's role in this phenomenon, remains in its infancy. Here, we discuss key work that has shaped our current understanding of these topics. Because tumour dormancy provides a unique therapeutic window to prevent metastatic disease, we discuss on-going clinical trials and weigh the potential for immunotherapy to eradicate dormant disease.

The Rodent Liver Undergoes Weaning-Induced Involution and Supports Breast Cancer Metastasis.

Patients with postpartum breast cancer are at increased risk for metastasis compared with age-matched nulliparous or pregnant patients. Here, we address whether circulating tumor cells have a metastatic advantage in the postpartum host and find the postlactation rodent liver preferentially supports metastasis. Upon weaning, we observed liver weight loss, hepatocyte apoptosis, extracellular matrix remodeling including deposition of collagen and tenascin-C, and myeloid cell influx, data consistent with weaning-induced liver involution and establishment of a prometastatic microenvironment. Using intracardiac and intraportal metastasis models, we observed increased liver metastasis in post-weaning BALB/c mice compared with nulliparous controls. Human relevance is suggested by a ∼3-fold increase in liver metastasis in patients with postpartum breast cancer (n = 564) and by liver-specific tropism (n = 117). In sum, our data reveal a previously unknown biology of the rodent liver, weaning-induced liver involution, which may provide insight into the increased liver metastasis and poor prognosis of women diagnosed with postpartum breast cancer. SIGNIFICANCE: We find that patients with postpartum breast cancer are at elevated risk for liver metastasis. We identify a previously unrecognized biology, namely weaning-induced liver involution, that establishes a prometastatic microenvironment, and which may account in part for the poor prognosis of patients with postpartum breast cancer. Cancer Discov; 7(2); 177-87. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115.

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 µl, ≥ 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of ≤ 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and > 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

Quantitative extracellular matrix proteomics to study mammary and liver tissue microenvironments.

Normal epithelium exists within a dynamic extracellular matrix (ECM) that is tuned to regulate tissue specific epithelial cell function. As such, ECM contributes to tissue homeostasis, differentiation, and disease, including cancer. Though it is now recognized that the functional unit of normal and transformed epithelium is the epithelial cell and its adjacent ECM, we lack a basic understanding of tissue-specific ECM composition and abundance, as well as how physiologic changes in ECM impact cancer risk and outcomes. While traditional proteomic techniques have advanced to robustly identify ECM proteins within tissues, methods to determine absolute abundance have lagged. Here, with a focus on tissues relevant to breast cancer, we utilize mass spectrometry methods optimized for absolute quantitative ECM analysis. Employing an extensive protein extraction and digestion method, combined with stable isotope labeled Quantitative conCATamer (QconCAT) peptides that serve as internal standards for absolute quantification of protein, we quantify 98 ECM, ECM-associated, and cellular proteins in a single analytical run. In rodent models, we applied this approach to the primary site of breast cancer, the normal mammary gland, as well as a common and particularly deadly site of breast cancer metastasis, the liver. We find that mammary gland and liver have distinct ECM abundance and relative composition. Further, we show mammary gland ECM abundance and relative compositions differ across the reproductive cycle, with the most dramatic changes occurring during the pro-tumorigenic window of weaning-induced involution. Combined, this work suggests ECM candidates for investigation of breast cancer progression and metastasis, particularly in postpartum breast cancers that are characterized by high metastatic rates. Finally, we suggest that with use of absolute quantitative ECM proteomics to characterize tissues of interest, it will be possible to reconstruct more relevant in vitro models to investigate tumor-ECM dynamics at higher resolution.

hTERT extends the life of human fibroblasts without compromising type I interferon signaling.

Primary cells are often used to study viral replication and host-virus interactions as their antiviral pathways have not been altered or inactivated; however, their use is restricted by their short lifespan. Conventional methods to extend the life of primary cultures typically utilize viral oncogenes. Many of these oncogenes, however, perturb or inactivate cellular antiviral pathways, including the interferon (IFN) response. It has been previously shown that expression of the telomerase reverse transcriptase (TERT) gene extends the life of certain cell types. The effect that TERT expression has on the innate antiviral response to RNA- and DNA-containing viruses has not been examined. In the current study, we introduced the human TERT (hTERT) gene into a primary human embryonic lung (HEL-299) cell strain, which is known to respond to the type I IFN, IFN-ß. We show that the resulting HEL-TERT cell line is capable of replicating beyond 100 population doublings without exhibiting signs of senescence. Treatment with IFN-ß resulted in the upregulation of four model IFN stimulated genes (ISGs) in HEL-299 and HEL-TERT cells. Both cell lines supported the replication of herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV) and impaired the replication of both viruses upon IFN-ß pretreatment. Introduction of the viral oncoprotein, simian virus 40 (SV40) large T-antigen, which is frequently used to immortalize cells, largely negated this effect. Taken together, our data indicate that expression of hTERT does not alter type 1 IFN signaling and/or the growth of two viruses, making this cell line a useful reagent for studying viral replication and virus-cell interactions.

CK2 inhibitors increase the sensitivity of HSV-1 to interferon-ß.

Herpes simplex virus type 1 (HSV-1) requires the activities of cellular kinases for efficient replication. The host kinase, CK2, has been shown or is predicted to modify several HSV-1 proteins and has been proposed to affect one or more steps in the viral life cycle. Furthermore, potential cellular and viral substrates of CK2 are involved in antiviral pathways and viral counter-defenses, respectively, suggesting that CK2 regulates these processes. Consequently, we tested whether pharmacological inhibitors of CK2 impaired HSV-1 replication, either alone or in combination with the cellular antiviral factor, interferon-ß (IFN-ß). Our results indicate that the use of CK2 inhibitors results in a minor reduction in HSV-1 replication but enhanced the inhibitory effect of IFN-ß on replication. This effect was dependent on the HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), which impairs several host antiviral responses, including that produced by IFN-ß. Inhibitors of CK2 did not, however, impede the ability of ICP0 to induce the degradation of two cellular targets: the promyelocytic leukemia protein (PML) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Notably, this effect was only apparent for HSV-1, as the CK2 inhibitors did not enhance the antiviral effect of IFN-ß on either vesicular stomatitis virus or adenovirus type 5. Thus, our data suggest that the activity of CK2 is required for an early function during viral infection that assists the growth of HSV-1 in IFN-ß-treated cells.