Breast Cancer Liver Metastases Research Articles

Identification of Genes Crucial for Biological Processes in Breast Cancer Liver Metastasis Relapse.

Breast cancer, when advancing to a metastatic stage, involves the liver, impacting over 50% of cases and significantly diminishing survival rates. Presently, a lack of tailored therapeutic protocols for breast cancer liver metastasis (BCLM) underscores the need for a deeper understanding of molecular patterns governing this complication. Therefore, by analyzing differentially expressed genes (DEGs) between primary breast tumors and BCLM lesions, we aimed to shed light on the diversities of this process. This research investigated breast cancer liver metastasis relapse by employing a comprehensive approach that integrated data filtering, gene ontology and KEGG pathway analysis, overall survival analysis, identification of the alteration in the DEGs, visualization of the protein-protein interaction network, Signor 2.0, identification of positively correlated genes, immune cell infiltration analysis, genetic alternation analysis, copy number variant analysis, gene-to-mRNA interaction, transcription factor analysis, molecular docking, and identification of potential treatment targets. This study's integrative approach unveiled metabolic reprogramming, suggesting altered PCK1 and LPL expression as key in breast cancer metastasis recurrence.

Abstract PO5-25-01: Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes

Abstract Background: Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates poor prognosis. Patients with BCLM have a 5-year survival rate of 8%. Specific treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must clear space among hepatocytes, the primary cell population in the liver, during metastatic formation. Cell competition is a developmentally conserved process that explains how organs or primary tumors form when cells within those tissues have varying fitness and compete with one another. This competition can result in the survival and expansion of one cell population, and the induction of cell death through apoptosis in the other. We hypothesized that MCC use cell competition with hepatocytes to create space and expand within the liver. Objectives: In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods: We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes, initially using balb/c derived breast cancer (4T1) and hepatocyte (H2.35) cells and syngeneic mouse models of liver metastasis. These models directly capture MCC and hepatocyte interactions, and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we next performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generated a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells. This dataset includes 3264 primary breast cancer cells across 3 models of aggressive breast cancers, encompassing HER2+ and TNBC subtypes, as well as 526 embryonic mammary gland cells. Using this platform, we identify genes enriched in both breast cancer and the developing mammary gland. Next, we validate genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determine the impact of target genes on cell competition in our models. Results/Discussion: Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice livers with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified 5 potential targets that could drive breast cancer metastasis and cell competition. Using our competition models, perturbation of SerpinE2, a protease inhibitor which was one of the top gene targets, successfully modulated competition between MCC and hepatocytes. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild type conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with wild-type (WT) MCC. These findings suggest that hepatocytes cannot outcompete or kill WT MCC, but they can outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Our findings suggest that restoring the competitiveness of the liver parenchyma could be a new treatment modality for treating liver metastases. Citation Format: Katherine Lake, Sakshi Mohta, Clayton Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Christine Zhang, Jacob Pena, Christine Hodgdon, Julia Maues, Elizabeth Chen, Isaac Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-01.

Outcome and Factors Related to Isolated Liver Metastasis due to Breast Cancer.

To determine the associated risk factors for isolated liver metastasis in breast cancer patients and to detect the prognostic factors related to survival. Analytical study. Place and Duration of the Study: Department of General Surgery, The University of Health Sciences, Istanbul, Turkiye, from January 2011 to November 2020. Patients with breast cancer liver metastasis who experienced surgery were retrospectively analysed for breast cancer and metastases-related characteristics. Descriptive statistical methods were used in the evaluation of data. Survival analyses were estimated by the Kaplan-Meier method. Log-rank and univariable Cox regression tests were utilised to search for prognostic factors' impact on survival. Out of 12 patients, 11 had recurrent disease after a median of 36 months of disease-free survival (DFS) and one patient had de novo metastasis. Grade 3 tumours and increased expression of Ki-67 had a negative effect on DFS. The median follow-up period was 66 months. Survival analysis showed 2- and 3-year progression-free survival (PFS); overall survival rates were 82%, 69%, 92%, and 82%, respectively. Development of liver metastasis in 3 years following breast cancer treatment was linked to worse PFS (p = 0.040). Long-term survival is possible for breast cancer survivors with liver metastasis. Disease-free interval is an important determinant. Longer progression-free survival was detected in patients who had developed metastasis after three years of breast cancer treatment. Breast cancer, Liver metastasis, Hepatic surgery.

CircLIFR-007 reduces liver metastasis via promoting hnRNPA1 nuclear export and YAP phosphorylation in breast cancer

Cancer metastasis is the major cause of death in patients with breast cancer (BC). The liver is a common site of breast cancer metastasis, and the 5-year survival rate of patients with breast cancer liver metastases (BCLMs) is only about 8.5 %. CircRNAs are involved in a variety of cancer-related pathological behaviors, and their unique structure and resistance to RNA degradation enable them to serve as ideal diagnostic biomarkers and therapeutic targets. Therefore, it is important to investigate the role and molecular mechanism of circRNAs in cancer metastasis. CircLIFR-007 was identified as a critical circular RNA in BC metastasis by circRNAs microarray and qRT-PCR experiment. Cell function assays were performed to explore the effect of circLIFR-007 in breast cancer cells. Experiments in vivo validated the function of circLIFR-007. Several molecular assays were performed to investigate the underlying mechanisms. We found that circLIFR-007 acted as a negative controller in breast cancer liver metastasis. CircLIFR-007 upregulates the phosphorylation level of YAP by exporting hnRNPA1 to promote the combination between hnRNPA1 and YAP in the cytoplasm. Overexpression of circLIFR-007 suppressed the expression of liver metastasis-related proteins, SREBF1 and SNAI1, which were regulated by transcription factor YAP. Functionally, circLIFR-007 inhibits the proliferation and metastasis of breast cancer cells both in vivo and in vitro.

Evaluation of markers of immunity in different metastatic immune microenvironments suggests more suppression within breast to liver metastases in breast cancer.

Programmed death receptor ligand-1 (PD-L1) expression and tumor mutational burden (TMB) are approved screening biomarkers for immune checkpoint inhibition (ICI) in advanced triple negative breast cancer. We examined these biomarkers along with characterization of the tumor microenvironment (TME) between breast tumors (BrTs), axillary metastases (AxMs), liver metastases (LvMs), non-axillary lymph node metastases, and non-liver metastases to determine differences related to site of metastatic disease. 3076 unpaired biopsies from breast cancer patients were analyzed using whole transcriptome sequencing and NextGen DNA depicting TMB within tumor sites. The PD-L1 positivity was determined with VENTANA PD-L1 (SP142) assay. The immune cell fraction within the TME was calculated by QuantiSeq and MCP-counter. Compared to BrT, more LvM samples had a high TMB (≥ 10 mutations/Mb) and fewer LvM samples had PD-L1+ expression. Evaluation of the TME revealed that LvM sites harbored lower infiltration of adaptive immune cells, such as CD4+, CD8+, and regulatory T-cells compared with the BrT foci. We saw differences in innate immune cell infiltration in LvM compared to BrT, including neutrophils and NK cells. LvMs are less likely to express PD-L1+ tumor cells but more likely to harbor high TMB as compared to BrTs. Unlike AxMs, LvMs represent a more immunosuppressed TME and demonstrate lower gene expression associated with adaptive immunity compared to BrTs. These findings suggest biopsy site be considered when interpreting results that influence ICI use for treatment and further investigation of immune composition and biomarkers expression by metastatic site.

Transcriptomic characterization of the histopathological growth patterns in breast cancer liver metastases.

Metastatic breast cancer (mBC) remains incurable and liver metastases (LM) are observed in approximately 50% of all patients with mBC. In some cases, surgical resection of breast cancer liver metastases (BCLM) is associated with prolonged survival. However, there are currently no validated marker to identify these patients. The interactions between the metastatic cancer cells and the liver microenvironment result in two main histopathological growth patterns (HGP): replacement (r-HGP), characterized by a direct contact between the cancer cells and the hepatocytes, and desmoplastic (d-HGP), in which a fibrous rim surrounds the tumor cells. In patients who underwent resection of BCLM, the r-HGP is associated with a worse postoperative prognosis than the d-HGP. Here, we aim at unraveling the biological differences between these HGP within ten patients presenting both HGP within the same metastasis. The transcriptomic analyses reveal overexpression of genes involved in cell cycle, DNA repair, vessel co-option and cell motility in r-HGP while angiogenesis, wound healing, and several immune processes were found overexpressed in d-HGP LM. Understanding the biology of the LM could open avenues to refine treatment of BC patients with LM.

Anthracycline-based hepatic arterial infusion chemotherapy achieved 17months of disease regression in a patient with breast cancer liver metastases resistant to multiple systemic chemotherapies.

Hepatic arterial infusion chemotherapy (HAIC) for liver metastases (LMs) from breast cancer is not a standard of care, but its effectiveness in patients with extensive LMs who cannot tolerate systemic therapy has been reported. Herein, we report a case of breast cancer LMs that were controlled by anthracycline-based HAIC. A 46-year-old woman with estrogen receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer who had multiple LMs and bone metastases underwent seven lines of systemic therapy (paclitaxel/bevacizumab for 38months; letrozole, nivolumab/fulvestrant, eribulin, gemcitabine/vinorelbine, high-dose toremifene/abemaciclib, and capecitabine for 21months in total). However, owing to its adverse effects and the continued progression of the LMs, systemic therapy was switched to HAIC (40mg/body epirubicin on day 1, 4mg/body mitomycin C on days 1 and 15, and 500mg/body 5-fluorouracil on days 1, 8, and 15; 28-day courses). In response to HAIC, the LMs remarkably regressed and were controlled for 17months without severe adverse effects. HAIC was stopped when multiple brain metastases arose, and the patient died 2months later. This case suggests that HAIC is a reasonable option for patients with extensive LMs, even in the late stage of treatment. HAIC recipients should be carefully selected through multidisciplinary discussions as the survival benefits of HAIC over systemic treatment remain unclear. Our findings identify a potential window for the use of traditional chemotherapeutic agents such as anthracyclines. Novel strategies to improve drug delivery are warranted in the future.

Abstract A041: Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes

Abstract Background Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates particularly poor prognosis. Targeted treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must first clear space among hepatocytes, the primary cell population in the liver, to form. Cell competition is a process that explains how organs or primary tumors form from two populations of competing cells with varied fitness levels. The expansion of one population of cells creates space through the induction of apoptosis in the other. Objectives In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes. These models directly capture MCC and hepatocyte interactions and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generate a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells and identify genes enriched in both. Next, we validated genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determined the impact of target genes on cell competition in our models. Results/Discussion Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified SerpinE2, a protease inhibitor, as a target that could drive breast cancer metastasis and cell competition. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild-type (WT) conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with WT MCC. These findings suggest that perturbation of SerpinE2 successfully modulates competition between MCC and hepatocytes: Hepatocytes can selectively outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Citation Format: Katherine E Lake, Sakshi Mohta, Clayton A Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Jacob Pena, Luise Chinea, Luisa Froessl, Sangeetha M Reddy, Heather L McArthur, Christine Hodges, Julia Maues, Elizabeth Chen, Isaac S Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A041.

Abstract A046: Liver involution generates a pro-metastatic niche in a murine model of postpartum breast cancer liver metastasis

Abstract Postpartum breast cancer (PPBC), cases diagnosed within 10 years of childbirth, has increased risk of liver metastases. Our lab has shown that the liver undergoes growth during pregnancy and lactation to support milk production, and upon weaning, undergoes involution. We have previously shown the actively involuting liver supports breast cancer liver metastasis in rodent models of PPBC, yet the mechanisms are poorly understood. Here, we performed RNAseq analysis in mouse livers across a lactation/wean cycle, and identify stromal alterations that may contribute to the “involution-educated” metastatic niche. During involution, RNA seq analysis reveals increased signatures for apoptotic cell death, catabolic metabolism, regulatory immune cell abundance, and extracellular matrix remodeling consistent with fibroblast activation. Using liver perfusion methods in live animals, we isolated viable stromal cells from livers of nullip or involution mice and validated these cells as highly enriched for fibroblasts by quantitative PCR. Liver fibroblasts were then mixed 1:1 with mouse mammary tumor cells, and injected into host mice at the flank site. We found fibroblasts from involuting livers generated larger tumors (n=28 tumors/group p=0.013), with decreased caspase 3 staining. Further, involution fibroblast tumors had increased ECM deposition as measured by trichrome staining, consistent with involution liver fibroblasts being more activated. Combined these data suggest that involution liver fibroblasts support tumor growth by suppressing tumor cell death, and by providing a pro-tumor collagen matrix when compared to nulliparous liver fibroblasts. These data implicate fibroblasts as components of the involution-educated metastatic niche in the postpartum liver. RNAseq analysis of involuting livers also identified gene signatures correlated with immature myeloid cells that associate with immune suppression and worse prognosis in numerous cancers. We thus evaluated for immature myeloid cells by multiplex immunohistochemistry in healthy mouse livers across a reproductive cycle, and identified increased abundance of immature myeloid cells (CD45+, CD11b+, Gr-1+) specifically during involution (n=6/group, p<0.05). Further, our data suggest liver involution may have a durable impact on immune composition of breast cancer liver metastases, as we find evidence for increased immature myeloid cells (CD45+, CD11b+, CD33+, CD68-, CD66b-, Cd56-, CD11c-, CD20-) in liver metastases of PPBC patients compared to non-PPBC patients. These data implicate immature myeloid cells as another component of the involution-educated liver metastatic niche. In sum, we find physiological involution of the liver post-wean alters the liver stromal microenvironment in a manner consistent with establishing a metastatic niche. Our findings may lead to the identification of liver stromal targets that can be exploited for prevention and or treatment strategies for PPBC liver metastasis. Citation Format: Michelle K Ozaki, Yi Zhang, Zheng Xi, Pepper Schedin. Liver involution generates a pro-metastatic niche in a murine model of postpartum breast cancer liver metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A046.

Abstract IA06: Postpartum breast involution informs breast cancer metastasis to the liver

Abstract Young women with breast cancer have disproportionally higher rates of metastasis, and effective treatment strategies are lacking. Our lab has focused on investigating how normal reproductive changes in tissue biology interface with breast cancer metastasis in young patients. These studies identify weaning-induced involution of the breast as a tissue remodeling process similar to wound healing that promotes metastasis in rodent models. Relevance to women is suggested by the fact that a breast cancer diagnosis within 5 years of childbirth is a strong and independent risk factor for metastasis. Further, these studies find that up to 50% of all young onset breast cancer is diagnosed within this high risk postpartum window. This subset of common, young onset breast cancer is referred to as postpartum breast cancer (PPBC). We find that PPBC appears durably altered by involution, as PPBC tumors display involution, metastatic, and treatment resistant molecular signatures. PPBC also specifically associates with increased liver metastases, a metastatic site associated with very poor prognosis. In rodents, we identified a functional link between the mammary gland and the liver, including weaning-induced liver involution. Liver involution is characterized by hepatocyte cell death, fibroblast activation, ECM deposition, and an immune regulatory infiltrate similar to that found in the postpartum involuting breast. In rodent models of breast cancer liver metastasis (BCLM), the involuting liver supports outgrowth of circulating breast cancer cells more than the nulliparous host liver. Evidence for similar liver biology occurring in women is suggested by studies in healthy women, where we find liver volume increase during pregnancy and loss post wean, data consistent with liver involution. In our mouse models of BCLM, the involution metastatic advantage coincided with weaning-induced immune tolerance in the liver. We next targeted the pro-tumor, immune suppressed state of involution with the immune modulatory agent vitamin D. These studies revealed dramatic down regulation of liver metabolism with weaning, including liver cytochrome P450 enzymes, which effectively nullified the protective effect of vitamin D in involution hosts. Combined, these novel findings are anticipated to have implications for sex-specific liver diseases broadly, efficacy of pro-drugs in the postpartum window, and for breast cancer outcomes in young patients. Citation Format: Pepper Schedin. Postpartum breast involution informs breast cancer metastasis to the liver [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA06.

Diagnostic yield of contrast-enhanced abdominal staging CT in patients with initially diagnosed breast cancer

PurposeTo estimate the diagnostic yield (DY) of abdominal staging CT for detecting breast cancer liver metastasis (BCLM) in patients with initially diagnosed breast cancer and to determine the indications for abdominal staging CT. MethodsPatients with newly diagnosed breast cancer who underwent abdominal CT as an initial staging work-up between January 2019 and December 2020 were retrospectively analyzed. DY was calculated and analyzed according to patient age, type of treatments, histologic type, histologic grade, lymphovascular invasion, Ki-67 status, hormone receptor status, subtype, and the American Joint Committee on Cancer anatomical staging. ResultsA total of 2056 patients (mean age, 51 ± 11 years) were included. The DY of abdominal staging CT for detecting BCLM was 1.1 % (22 of 2056). DY was significantly higher in stage III than in stage I or II cancers (3.9 % [18 of 467] vs. 0 % [0 of 412] or 0.4 % [4 of 1158], respectively, p < .001), and in human epidermal growth factor receptor-2 (HER2)-enriched cancers than in luminal or triple negative cancers (2.9 % [16 of 560] vs. 0.4 % [4 of 1090] or 0.5 % [2 of 406], respectively, p < .001). ConclusionsThe DY of abdominal staging CT for detecting BCLM was low among all patients with initially diagnosed breast cancer. However, although abdominal staging CT for detecting BCLM is probably unnecessary in all patients, it can be clinically useful in patients with stage III or human epidermal growth factor receptor-2-enriched breast cancers.

Histopathological growth patterns and tumor-infiltrating lymphocytes in breast cancer liver metastases

Liver is the third most common organ for breast cancer (BC) metastasis. Two main histopathological growth patterns (HGP) exist in liver metastases (LM): desmoplastic and replacement. Although a reduced immunotherapy efficacy is reported in patients with LM, tumor-infiltrating lymphocytes (TIL) have not yet been investigated in BCLM. Here, we evaluate the distribution of the HGP and TIL in BCLM, and their association with clinicopathological variables and survival. We collect samples from surgically resected BCLM (n = 133 patients, 568 H&E sections) and post-mortem derived BCLM (n = 23 patients, 97 H&E sections). HGP is assessed as the proportion of tumor liver interface and categorized as pure-replacement (‘pure r-HGP’) or any-desmoplastic (‘any d-HGP’). We score the TIL according to LM-specific guidelines. Associations with progression-free (PFS) and overall survival (OS) are assessed using Cox regressions. We observe a higher prevalence of ‘any d-HGP’ (56%) in the surgical samples and a higher prevalence of ‘pure r-HGP’ (83%) in the post-mortem samples. In the surgical cohort, no evidence of the association between HGP and clinicopathological characteristics is observed except with the laterality of the primary tumor (p value = 0.049) and the systemic preoperative treatment before liver surgery (p value = .039). TIL is less prevalent in ‘pure r-HGP’ as compared to ‘any d-HGP’ (p value = 0.001). ‘Pure r-HGP’ predicts worse PFS (HR: 2.65; CI: (1.45–4.82); p value = 0.001) and OS (HR: 3.10; CI: (1.29–7.46); p value = 0.011) in the multivariable analyses. To conclude, we demonstrate that BCLM with a ‘pure r-HGP’ is associated with less TIL and with the worse outcome when compared with BCLM with ‘any d-HGP’. These findings suggest that HGP could be considered to refine treatment approaches.

Euphorbia factorL2 suppresses the generation of liver metastatic ascites in breast cancer via inhibiting NLRP3 inflammasome activation.

Metastasis is the leading cause of death in patients with breast cancer, in part due to the lack of effective treatments. Euphorbia factorL2 (EFL2) is a diterpenoid extracted from Euphorbia lathyris L. seeds, which has attracted increasing attention in recent years due to its anticancer effect. However, the role and molecular mechanism of EFL2 in breast cancer liver metastasis remain unclear. In the present study, a breast cancer liver metastasis model was constructed and the effect of EFL2 on ascites generation in mice was examined. H&E staining detected inflammatory cells and tumor cells in the liver, small intestine and tumor tissues. Western blotting and reverse transcription‑quantitative PCR were used to detect the protein and mRNA expression of NLR family pyrin domain containing‑3 (NLRP3) and related molecules in tumor tissues. Immunohistochemistry was used to detect the levels of CD4 and CD8 Tcells in tumor tissue and immunofluorescence was used to further detect the expression level of NLRP3. Finally, the aforementioned experiments were further verified by overexpressing NLPR3. It was found that EFL2 inhibited generation of ascites in the model in a dose‑dependent manner. Furthermore, EFL2 inhibited tumor cell metastasis and enhanced immune cell infiltration. Meanwhile, EFL2 dose‑dependently downregulated the mRNA and protein expression of NLRP3 and related molecules in the model, and overexpression of NLRP3 abolished these beneficial effects of EFL2. Taken together, the present experimental data suggested that EFL2 has a significant inhibitory effect on ascites of breast cancer liver metastasis invivo, which may inhibit tumor cell metastasis by downregulating NLRP3 expression, providing an experimental basis for treating breast cancer metastasis.

Interventional oncology in breast cancer.

Breast cancer (BC) is the most common cancer and one of the most important causes of death in women. Surgery is the standard therapy for breast cancer and in the last decades evolved towards a more conservative approach, with lumpectomy, followed by radiation therapy, the most common option. Unfortunately, up to 40% of women affected by BC will develop metastases and will receive systemic therapy, which improves survival and quality of life. Interventional oncology (IO), thanks to the improvement in technology and clinical experience, is gaining an important role in the field of breast cancer, both in treating the primary tumour and also in metastasis in well-selected cases. Percutaneous thermal ablation and more recently cryoablation are reported to achieve promising results in the radical treatment of small breast cancer, with optimal cosmetic outcome and a very high safety profile. Percutaneous ablation as well as intra-arterial therapies, such as chemoembolization and radioembolization, might also be indicated in metastatic BC patients. In advanced stage disease, breast cancer liver metastases (BCLM) represent the main factor affecting the overall survival. Metastatic breast disease is a systemic disease, with tumour deposits potentially spread into different organs and tissues for which systemic therapy is the standard approach. Local therapies for liver metastases might have an important role in improving survival and quality of life in well-selected patients. Clinical and technical indications with their limitations, results and potential complications in local IO treatment for BCLM, will be also described.

Euphorbia factor L1 suppresses breast cancer liver metastasis via DDR1-mediated immune infiltration.

Euphorbia factor L1 (EFL1), a lathyrane-type diterpenoid from the medicinal herb Euphorbia lathyris L., has been documented to possess various pharmacologic actives. However, the function of EFL1 on breast cancer is not clear. In this study, we explored the effect and mechanism of EFL1 on breast cancer liver metastasis. Female BALB/c mice were subjected to breast cancer-surgical hepatic implantation (SHI) to establish breast cancer liver metastasis model in vivo. At 10 days post-surgery, mice were administrated with EFL1 once daily for a total of 2 weeks. Serum AST and ALT activities, abdominal circumference, peritoneal fluid, tumor weight and volume were determined to assess liver and mesenteric re-metastasis of breast cancer. H&E staining was used to observe morphology changes in tumor, liver and small intestine tissues. ELISA was applied to observe inflammatory levels. Tumor DDR1 expression and immune infiltration were determined using western blotting, immunohistochemistry and flow cytometer methods. Our results showed that EFL1 administration improved liver function (AST and ALT activities), ascites, liver metastasis and mesenteric re-metastasis in SHI mice. Also, SHI-induced inflammatory cell infiltration and IL-1β, IL-6, TNF-α generation in ascites were decreased by EFL1 treatment. Mechanism study revealed that EFL1 intervention enhanced the ratios of CD4+ and CD8+ and CD49b+(NK) T lymphocytes and decreased Treg cells through downregulating DDR1 in the tumor of SHI mice. Furthermore, overexpression of DDR1 abolished the anti-liver metastasis effect and pro-immune infiltration action of EFL1 in SHI mice. Together, our findings suggested that EFL1 protects against breast cancer liver metastasis in vivo by targeting DDR1-mediated immune infiltration.

Receptor conversion and survival in breast cancer liver metastases

BackgroundBreast cancer liver metastases (BCLM) is a common cause of breast cancer-related death. The prognostic and predictive value of receptor expression and St Gallen classification is challenged by receptor status discordance in distant metastases. The aim of this study was to determine the rate of receptor conversion from breast cancer to BCLM and the impact on survival.MethodPatients registered with BCLM in two Swedish national cancer registers were recruited retrospectively. Data on receptor expression in primary breast cancer and BCLM were collected, as well as information about predictive factors for survival. The rate of receptor and subtype conversion was analyzed. A Cox regression model was used to investigate predictive factors for survival.ResultsA cohort of 132 patients with BCLM was identified. Estrogen receptor (ER), progesterone receptor (PgR) and HER2 converted in 17, 33 and 10%, respectively. PgR was lost in BCLM while 8/10 HER2 conversions went from negative to positive. The BC subtype was re-classified in 21% of the BCLM. Median survival after BCLM was 13 months and HER2 amplification was associated with improved survival (HR 0.28 CI 0.085–0.90). The highest predictive value (Harrell´s C-index) was obtained when including both BC and BCLM status.ConclusionsReceptor and subtype conversions are common in BCLM, and a liver biopsy is warranted to tailor BCLM treatment. HER2 amplification is associated with improved survival in a BCLM cohort.

A gene mutation-based risk model for prognostic prediction in liver metastases

BackgroundLiver metastasis is the major challenge in the treatment for malignant tumors. Genomic profiling is increasingly used in the diagnosis, treatment and prediction of prognosis in malignancies. In this study, we constructed a gene mutation-based risk model to predict the survival of liver metastases.MethodWe identified the gene mutations associated with survival and constructed the risk model in the training cohort including 800 patients with liver metastases from Memorial Sloan-Kettering Cancer Center (MSKCC) dataset. Other 794 patients with liver metastases were collected from 4 cohorts for validation. Furthermore, the analyses of tumor microenvironment (TME) and somatic mutations were performed on 51 patients with breast cancer liver metastases (BCLM) who had both somatic mutation data and RNA-sequencing data.ResultsA gene mutation-based risk model involved 10 genes was constructed to divide patients with liver metastases into the high- and low-risk groups. Patients in the low-risk group had a longer survival time compared to those in the high-risk group, which was observed in both training and validation cohorts. The analyses of TME in BCLM showed that the low-risk group exhibited more immune infiltration than the high-risk group. Furthermore, the mutation signatures of the high-risk group were completely different from those of the low-risk group in patients with BCLM.ConclusionsThe gene mutation-based risk model constructed in our study exhibited the reliable ability of predicting the prognosis in liver metastases. The difference of TME and somatic mutations among BCLM patients with different risk score can guide the further research and treatment decisions for liver metastases.

Gut colonization with an obesity-associated enteropathogenic microbe modulates the premetastatic niches to promote breast cancer lung and liver metastasis.

Obesity, an independent risk factor for breast cancer growth and metastatic progression, is also closely intertwined with gut dysbiosis; and both obese state and dysbiosis promote each other. Enteric abundance of Bacteroides fragilis is strongly linked with obesity, and we recently discovered the presence of B. fragilis in malignant breast cancer. Given that enterotoxigenic B. fragilis or ETBF, which secretes B. fragilis toxin (BFT), has been identified as a procarcinogenic microbe in breast cancer, it is necessary to examine its impact on distant metastasis and underlying systemic and localized alterations promoting metastatic progression of breast cancer. We used syngeneic mammary intraductal (MIND) model harboring gut colonization with ETBF to query distant metastasis of breast cancer cells. Alterations in the immune network and cytokines/chemokines in the tumor microenvironment and distant metastatic sites were examined using flow cytometry, immunohistochemistry, and multiplex arrays. ETBF infection initiates a systemic inflammation aiding in the establishment of the premetastatic niche formation in vital organs via increased proinflammatory and protumorigenic cytokines like IL17A, IL17E, IL27p28, IL17A/F, IL6, and IL10 in addition to creating a prometastatic immunosuppressive environment in the liver and lungs rich in myeloid cells, macrophages, and T regulatory cells. It induces remodeling of the tumor microenvironment via immune cell and stroma infiltration, increased vasculogenesis, and an EMT-like response, thereby encouraging early metastatic dissemination ready to colonize the conducive environment in liver and lungs of the breast tumor-bearing mice. In this study, we show that enteric ETBF infection concomitantly induces systemic inflammation, reshapes the tumor immune microenvironment, and creates conducive metastatic niches to potentiate early dissemination and seeding of metastases to liver and lung tissues in agreement with the "seed and soil hypothesis." Our results also support the ETBF-induced "parallel model" of metastasis that advocates for an early dissemination of tumor cells that form metastatic lesions independent of the primary tumor load.

The mechanism of exosome delivered circ-PDE7B promoting liver metastasis of breast cancer.

e13016 Background: According to “seed and soil” hypothesis, tumor microenvironments have major effects on the survival and metastasis of cancers, including breast cancer. Importantly, exosomes are believed to partake in cellular communications through delivering oncogenic ncRNAs (including circRNAs) to the recipient cells and tumor microenvironment, which resulting in distant metastasis. Unfortunately, exosomes delivering circRNAs from breast cancer cells still remained many potential functions that are not completely understood in the progression of liver metastasis of breast cancer. This study aims to provide a theoretical basis to use stable exosomal circRNAs as new biomarkers and target spot for the liver metastasis of breast cancer. Methods: A strategy combining RNA-sequencing technique, RT-qPCR and bioinformatic analysis were used to determine the level of differential expressed circRNAs in tissue exosomes (n=5) and serum exosomes samples (n=5) from liver metastatic breast cancer patients compared with healthy person. The expression of circPDE7B was also detected in tumor tissues (n=10) from liver metastatic patients compared with healthy person, breast cancer cell lines (MDA-MB-231), high-liver metastatic cell lines (LM-MDA-MB-231), exosome from MDA-MB-231 (231/exo) and from LM-MDA-MB-231 (LM-231/exo) by RT-qPCR. In animal experiments, exosomes (enriched in circPDE7B) were injected from tail vein for 24h, follow up with injection with LM-MDA-MB-231 cells, then, fluorescence imaging and pathology showed the metastasis in liver. Results: Isolated serum exosomes were characterization by transmission electron microscope (TEM) and Nanosight 300 (NTA 300) system. We profiled the circRNAs in the tissue and serum exosomes samples from liver metastatic patients and healthy person by RNA sequencing and detected 332 significantly differentially-expressed circRNAs. After bioinformatic analysis with GEO database, circPDE7B was chose to further study. RT-qPCR results showed that higher expression of circPDE7B in liver metastatic tissue and LM-MDA-MB-231, also in LM-231/exo. Moreover, overexpressed circPDE7B in exosomes from breast cancer changes premetastatic microenvironment of the liver microenvironment, which can promote the occurrence of breast cancer liver metastasis. Conclusions: The expression of circPDE7B was found significantly upregulated in tissue and serum exosomes from liver metastatic breast cancer patients. Moreover, circPDE7B could promote liver metastasis of breast cancer. Therefore, this study provides a fresh perspective on novel therapeutic targets and potential biomarkers for liver metastasis from exosomal circRNAs.

Risk factors of early liver metastasis in patients with breast cancer.

e13011 Background: Breast Cancer Liver Metastasis is very common in the clinical treatment of breast cancer. Although great progress has been made in the early diagnosis and treatment of breast cancer, 20% to 30% of patients with early breast cancer will develop tumor metastasis. Relevant studies have found that liver metastasis is an independent predictor of poor overall survival in breast cancer patients compared with bone metastasis and other factors. There is a lack of large-scale clinical research on the risk factors of early liver metastasis of breast cancer across the world. This study aims to explore the possible risk factors of early liver metastasis of breast cancer patients by analyzing the clinical data of 70 breast cancer patients with first liver metastasis. Methods: A retrospective study was conducted on 70 female breast cancer patients with liver metastasis who were diagnosed and treated in the First Affiliated Hospital of Nanjing Medical University from January 2011 to December 2021 and had complete medical records. The main clinical and pathological data of the patients were obtained by reading the medical records, such as age, menstrual status, surgical method, history of non-alcoholic fatty liver disease, T stage of the primary tumor, molecular type, vascular tumor thrombus and pathological type. SPSS 25.0 statistical software was used to analyze the data. Kaplan-Meier Survival curve and log-rank test were used to compare the disease-free survival (DFS) of breast cancer patients with and without non-alcoholic fatty liver Disease, different ER expression, Ki67 expression and histological grade. DFS is the interval between breast cancer surgery and the occurrence of metastases. COX regression model was used to analyze the risk factors of early metastasis of breast cancer. P &lt; 0.05 was considered statistically significant. Results: The median liver metastasis-free time of the 70 breast cancer patients was 34 months (range 4-121 months). High histological grade, non-alcoholic fatty liver disease, negative expression of ER and Ki-67&gt;15% were risk factors for early liver metastasis in patients with breast cancer after operation (χ2 =8.367, P=0.015; χ2 =6.883, P=0.009; χ2 =7.507, P=0.006; χ2 =10.488, P=0.001). Conclusions: In this study, the independent risk factors of early liver metastasis in breast cancer patients are high histological grade, high expression of Ki-67, and combined with alcoholic fatty liver, which is consistent with most studies at home and abroad, and proves that Chinese breast cancer patients have the same risk factors as foreign patients. However, the role of ER, PR and HER-2 expression is unclear, which may be related to the fact that some patients do not receive systemic endocrine and targeted therapy. More samples and multi-center prospective clinical studies are needed to confirm, but it still has certain clinical significance for the prognosis and intervention of breast cancer patients.