Model Of Breast Cancer Metastasis Research Articles

Proanthocyanidins attenuate breast cancer-induced bone metastasis by inhibiting Irf-3/c-jun activation.

We have previously demonstrated the pivotal role of Jnk-mediated Irf-3/c-Jun in regulating nuclear factor kappa-Β ligand (RANKL)-induced osteoclastogenesis. Here, we demonstrated that proanthocyanidins (PACs) target Irf-3 to alleviate breast cancer-induced activation of osteoclasts. We also found that PACs induced apoptosis of osteoclast precursors by upregulating the ratio of bax/bcl-2 and activating caspase-3 activity. Such bone protective effect also could be observed in a bone metastasis model of breast cancer. These findings provided a novel therapeutic intervention targeting abnormal bone metabolism to alleviate bone metastasis of breast cancer.

TGLI1 transcription factor mediates breast cancer brain metastasis via activating metastasis-initiating cancer stem cells and astrocytes in the tumor microenvironment.

Mechanisms for breast cancer metastasis remain unclear. Whether truncated glioma-associated oncogene homolog 1 (TGLI1), a transcription factor known to promote angiogenesis, migration and invasion, plays any role in metastasis of any tumor type has never been investigated. In this study, results of two mouse models of breast cancer metastasis showed that ectopic expression of TGLI1, but not GLI1, promoted preferential metastasis to the brain. Conversely, selective TGLI1 knockdown using antisense oligonucleotides led to decreased breast cancer brain metastasis (BCBM) in vivo. Immunohistochemical staining showed that TGLI1, but not GLI1, was increased in lymph node metastases compared to matched primary tumors, and that TGLI1 was expressed at higher levels in BCBM specimens compared to primary tumors. TGLI1 activation is associated with a shortened time to develop BCBM and enriched in HER2-enriched and triple-negative breast cancers. Radioresistant BCBM cell lines and specimens expressed higher levels of TGLI1, but not GLI1, than radiosensitive counterparts. Since cancer stem cells (CSCs) are radioresistant and metastasis-initiating cells, we examined TGLI1 for its involvement in breast CSCs and found TGLI1 to transcriptionally activate stemness genes CD44, Nanog, Sox2, and OCT4 leading to CSC renewal, and TGLI1 outcompetes with GLI1 for binding to target promoters. We next examined whether astrocyte-priming underlies TGLI1-mediated brain tropism and found that TGLI1-positive CSCs strongly activated and interacted with astrocytes in vitro and in vivo. These findings demonstrate, for the first time, that TGLI1 mediates breast cancer metastasis to the brain, in part, through promoting metastasis-initiating CSCs and activating astrocytes in BCBM microenvironment.

Effective treatment of cancer metastasis using a dual-ligand nanoparticle.

Metastasis is responsible for the majority of deaths of breast cancer patients. While cytotoxic drugs are available with high potency to kill breast cancer cells, they are not designed to specifically seek and navigate in the dynamic and continuously changing microenvironment of metastatic disease. To effectively delivery chemotherapeutic agents to metastasis, we designed a dual-ligand nanoparticle loaded with doxorubicin by using two different types of ligands targeting EGFR and αvβ3 integrin. Metastatic cancer cells continuously change resulting in heterogeneity even across adjacent micrometastatic regions with variable expression of these targetable receptors. Using a mouse model of breast cancer metastasis, in vivo and ex vivo imaging showed that both EGFR and αvβ3 integrin-targeting were required to reliably direct the nanoparticle to metastasis and capture the spread and exact topology of the disease. Survival studies compared the anticancer efficacy of the standard drug, EGFR-targeting nanoparticle, αvβ3 integrin-targeting nanoparticle and the dual-ligand nanoparticle. While all the other treatments produced moderate therapeutic outcomes, treatment with the dual-ligand nanoparticle yielded significant improvement and event-free survival in a mouse model of breast cancer metastasis.

Abstract 4500: Mechanisms of tumor-associated myeloid cells in modulating host immune microenvironment and metastatic progression

Abstract The immune checkpoint inhibitor blockade takes advantage of the specific receptor-ligand interactions between immune and tumor cells. However, the immunosuppressive microenvironment has been largely ignored, which likely contribute to low efficacy and resistance in immune therapies. Our studies demonstrate that TGF-β signaling in tumor-associated myeloid cells isessential in systemic immune suppression and metastatic progression. Using mouse models of breast cancer metastasis, we further uncover that miR130a and 145 are critical in regulating TGF-β signaling in myeloid cells. miR130a and 145 are down-regulated in these myeloid cells, leading to increased TGF-β RII. Ectopic expression of miR-130a and miR-145 in the myeloid cells decrease tumor metastasis. This is mediated through a downregulation of type 2 cytokines in myeloid cells and an increase in IFNgamma-producing cytotoxic CD8 T lymphocytes. Lastly, miR-130a and miR-145 mimics improve systemic anti-tumor immunity and inhibit metastasis in preclinical mouse models. Another important mechanism in myeloid-mediated immune suppression involves a decreased expression of Lamin A/C, nuclear lamina proteins that prominently interact with heterochromatin. Lamin A/C knockout in myeloid cells leads to H3K4me3-mediated upregulation of Gfi1 and C/EBPϵ,critical transcription factors responsible for granulocytic lineage differentiation. In addition, loss of Lamin A/C attenuates antigen-presentation and compromises host anti-tumor immunity. These mechanistic understandings provide options to reprogram tumor-associated myeloid cells by altering the cytokine milieu and metastatic microenvironment, thus enhancing host antitumor immunity. Citation Format: Hiroki Ishii, Christine Hollander, Tim Greten, Li Yang. Mechanisms of tumor-associated myeloid cells in modulating host immune microenvironment and metastatic progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4500.

Abstract 4390: Dietary soy isoflavone equol promotes breast cancer progression via regulation of protein synthesis initiation

Abstract We previously reported that dietary daidzein, a soy isoflavone, increased breast cancer progression in a nude mouse model of breast cancer metastasis. Further studies indicated that equol, a metabolite of the soy isoflavone daidzein is responsible for the cancer promoting effects of soy isoflavones in breast cancer cells in vitro. We reported that equol increases the expression of the eukaryotic protein synthesis initiation factor eIF4G1, and the transcription factor c-Myc. Increased eIF4G in response to equol resulted in the non-canonical protein synthesis of pro-cancer molecules such as Cyclin D, Bcl-XL, p120, matrix metalloproteinases, etc., as confirmed by ribosome profiling and eIF4G1 knockdown studies. This study tested the hypothesis that equol promoted breast cancer progression via eIF4G1 upregulation. Immunocompromised nude mice were used to establish mammary fatpad tumors from human metastatic breast cancer cells expressing eIF4G1 knockdowns via a Tetracycline-inducible promoter. Oral gavage of 50 mg/kg BW Doxycycline 3X a week for 4 weeks was enough to knockdown eIF4G1 expression by ~80%. Therefore, one week following tumor establishment, the mice were treated with vehicle (90% corn oil, 10% ethanol), equol, Doxycycline or, equol and Doxycycline. Data show that as expected, equol treatment increased tumor growth relative to vehicle treatment. Moreover, eIF4G1 knockdown abolished the effect of equol on tumor growth. Interestingly, the effect of eIF4G1 knockdown on mammary tumor growth was not evident until 55 days of doxycycline treatment. Therefore, we tested the cell and tumor lysates for potential expression of the eIF4G1 isoform Dap5 when eIF4G1 is knocked out. Dap5 protein was expressed in eIF4G1 knockdown cells and was increased by equol treatment, indicating that Dap5 may compensate for eIF4G1 knockdown by inducing alternative cap-mediated protein synthesis. In conclusion, the cancer promoting effect of equol is abolished in breast tumors where eIF4G1 expression was stably knocked down, thus, validating our hypothesis that equol increases breast cancer progression via upregulation of eIF4G1 dependent protein synthesis initiation. Citation Format: Ailed M. Cruz-Collazo, Columba De la Parra, Robert Schneider, Suranganie Dharmawardhane. Dietary soy isoflavone equol promotes breast cancer progression via regulation of protein synthesis initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4390.

Lymph-circulating tumor cells show distinct properties to blood-circulating tumor cells and are efficient metastatic precursors.

The leading cause of breast cancer‐associated death is metastasis. In 80% of solid tumors, metastasis via the lymphatic system precedes metastasis via the vascular system. However, the molecular properties of tumor cells as they exit the primary tumor into the afferent lymphatics en route to the sentinel lymph nodes (SLNs) are not yet known. Here, we developed an innovative technique that enables the collection of lymph and lymph‐circulating tumor cells (LCTCs) en route to the SLN in an immunocompetent animal model of breast cancer metastasis. We found that the gene and protein expression profiles of LCTCs and blood‐circulating tumor cells (BCTCs) as they exit the primary tumor are similar, but distinct from those of primary tumors and lymph node metastases (LNMs). LCTCs, but not BCTCs, exist in clusters, display a hybrid epithelial/mesenchymal phenotype and cancer stem cell‐like properties, and are efficient metastatic precursors. These results demonstrate that tumor cells that metastasize through the lymphatic system are different from those spread by blood circulation. Understanding the relative contribution of these cells to overall peripheral blood‐circulating tumor cells is important for cancer therapy. Whether these two types of cell occur in cancer patients remains to be determined.

Endogenous Production of IL1B by Breast Cancer Cells Drives Metastasis and Colonization of the Bone Microenvironment.

Breast cancer bone metastases are incurable, highlighting the need for new therapeutic targets. After colonizing bone, breast cancer cells remain dormant, until signals from the microenvironment stimulate outgrowth into overt metastases. Here we show that endogenous production of IL1B by tumor cells drives metastasis and growth in bone. Tumor/stromal IL1B and IL1 receptor 1 (IL1R1) expression was assessed in patient samples and effects of the IL1R antagonist, Anakinra, or the IL1B antibody canakinumab on tumor growth and spontaneous metastasis were measured in a humanized mouse model of breast cancer bone metastasis. Effects of tumor cell-derived IL1B on bone colonization and parameters associated with metastasis were measured in MDA-MB-231, MCF7, and T47D cells transfected with IL1B/control. In tissue samples from >1,300 patients with stage II/III breast cancer, IL1B in tumor cells correlated with relapse in bone (HR = 1.85; 95% CI, 1.05-3.26; P = 0.02) and other sites (HR = 2.09; 95% CI, 1.26-3.48; P = 0.0016). In a humanized model of spontaneous breast cancer metastasis to bone, Anakinra or canakinumab reduced metastasis and reduced the number of tumor cells shed into the circulation. Production of IL1B by tumor cells promoted epithelial-to-mesenchymal transition (altered E-Cadherin, N-Cadherin, and G-Catenin), invasion, migration, and bone colonization. Contact between tumor and osteoblasts or bone marrow cells increased IL1B secretion from all three cell types. IL1B alone did not stimulate tumor cell proliferation. Instead, IL1B caused expansion of the bone metastatic niche leading to tumor proliferation. Pharmacologic inhibition of IL1B has potential as a novel treatment for breast cancer metastasis.

FKBPL and its peptide derivatives inhibit endocrine therapy resistant cancer stem cells and breast cancer metastasis by downregulating DLL4 and Notch4

BackgroundOptimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease.MethodsIn this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett’s multiple comparison test.ResultsWe demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4.ConclusionThis study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.

Tumor Necrosis Factor-α Produced by Osteoclasts Might Induce Intractable Pain in a Rat Spinal Metastasis Model of Breast Cancer.

Introduction Causes of pain due to spinal metastases have been insufficiently investigated. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were the focus of this study. Both are known as proinflammatory cytokines associated with the pathophysiology of pain syndromes1). It is well known that cancer cells produce these cytokines, but whether osteoclasts produce them as well remains unclear. We hypothesize that osteoclasts produce these cytokines; in other words, pain from spinal metastasis is stronger than pain from the primary tumor. Methods We made a rat spinal metastasis model of breast cancer (metastasis group) and models with a hole in the vertebrae (puncture group) and resected the vertebrae. Tartrate-resistant acid phosphatase (TRAP) staining was performed to reconfirm that osteoclasts increase in vertebrae with spinal metastasis. We then evaluated TNF-α and IL-6 expression using immunohistochemistry and real-time polymerase chain reaction (PCR). Results The results of TRAP staining showed that osteoclasts increase in metastatic vertebrae. The osteoclasts in the puncture models were TNF-α negative but were TNF-α positive in the metastasis model. The osteoclasts in the puncture models and metastasis model were both IL-6 positive. According to the real-time PCR results, TNF-α in vertebrae increased in the metastasis model, but IL-6 did not increase in the metastasis model compared with in the puncture model. Conclusions The number of osteoclasts is higher in the metastasis model. While TNF in the osteoclasts increased in the spinal metastasis model, IL-6 did not. This probably means that breast cancer affects TNF production in osteoclasts. This increase of TNF-α may lead to pain from spinal metastasis.

Relationship between one-stop CT spectral perfusion imaging parameters and expression of lymphatic microvessel density and vascular endothelial growth factor-C in axillary lymph nodes of rabbit VX2 breast cancer

Objective: To analyze the correlation between quantitative parameters of one-stop spectral perfusion computed tomography imaging and the expression level of vascular endothelial growth factor-C(VEGF-C) and MLVD in metastatic lymph nodes of rabbit VX2 breast cancer. Methods: Thirty New Zealand purebred female rabbits were used to establish the lymph node metastasis model of rabbit VX2 breast cancer, one-stop spectral and perfusion CT imaging protocol was performed.The axillary lymph nodes were selected for corresponding image markers. The observed morphology of conventional HE staining and the EnVision method were used to quantitatively analyze VEGF-C expression and calculate MLVD.Pearson linear correlation was used to analyze the perfusion parameters of metastatic lymph node energy spectrum and the correlation of MLVD and VEGF-C expression. Results: Twenty-four experimental rabbits were successfully modeled and performed a one-stop CT scan on the 28th day. A total of 39 metastatic lymph nodes were included. The VEGF-C of metastatic lymph node was 20.0%±2.8%,and the MLVD was 12.5±3.5. There was a positive correlation between BF, AP(λHU), IC(VP), NIC(VP), VP(λHU) of metastatic lymph node and VEGF-C and MLVD (P<0.05). There was a positive correlation between lymph node IC(AP) and MLVD (P=0.027) and no correlation with VEGF-C expression (P=0.386).There was no correlation between BV, NIC(A)P and VEGF-C, MLVD(P>0.05). The correlation between VP(λHU) and MLVD was higher (r=0.448, P=0.001). Conclusions: One-stop CT spectral perfusion imaging quantitative parameters and pathological indicators have a good correlation, and it can reflect lymphatic vessel metastasis in lymph nodes.

Modeling Tumor Development, Progression and Metastasis in 3D Artificial Tissues: Characterization of Structure and Labeling Patterns in Breast Cancer Hanging‐drop Spheroid Cultures

In this report our laboratory presents the results of a series of studies being conducted to explore the application of 3D artificial tumor tissues as in‐vitro models of breast cancer development, progression and metastasis. In this case, we have employed MCF‐7 breast ductal adenocarcinoma cells and 3D Biomatrix hanging drop plates to evaluate these tumor properties within the context of staged tumor spheroids. A consistent observation of ours in nearly a decade of modeling breast cancer using various natural 3D scaffolds and hydrogel matrices has been that solid tumor models grown in long‐term culture predictably begin to shed both single cells and cell clusters in the form of small groupings of cells as well as progressively larger spheroids. Although we have shown that at least some of these structures have properties consistent with metastasis, including attachment and invasion of target tissues, spontaneously generated versions of cancer clusters also present as pleiomorphic structures and in relatively small numbers which complicates their study. Therefore, we sought to use a system that generates identical clusters and spheres from specific numbers of suspended single cells. The hanging drop system we employ comes in a 96 well format that allows us to generate literally as many spheres as we need for each study. Furthermore, these spheres can be grown out to match any of the sizes commonly found in our tumor models thereby allowing us to study each stage for structure, cell populations and markers. In addition these spheres are also assisting us in systematically studying the process of cancer cell cluster attachment and invasion and to test our working hypothesis that this is controlled through activation of EMT‐like responses through CD44 binding of hyaluronate on the target tissue. Using direct labeling of intact spheres coupled to embedding and sectioning followed by immunolabeling, these studies are demonstrating that over a period of 144 hours our spheroid cultures progress from simple clusters of a few cells to aggregated spheres containing thousands of cells and measuring up to ~600um in diameter. By this later stage, many spheres actually begin to generate their own smaller spheres and clusters as well. In our view this reflects the concept that past 48 hours in culture these spheres transition into being actual tumor organoids themselves. This is supported by development of a complex CD24+ internal structure distinct from an outer shell of CD44+ cells. These studies are ongoing and support our view that 3D artificial tumors are effective tools to study cancer biology in‐vitro.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PC-2 A liposomal formulation of HIF-1α inhibitor echinomycin inhibits growth and metastasis of experimental model of breast cancer

PC-2 A liposomal formulation of HIF-1α inhibitor echinomycin inhibits growth and metastasis of experimental model of breast cancer

Does the mouse tail vein injection method provide a good model of lung cancer?

Lung cancer drug development requires screening in animal models. We aimed to develop orthotopic models of human non-small lung cancer using A549 and H3122 cells delivered by tail vein injection. This procedure has been used previously for a mouse lung cancer (Lewis lung carcinoma) and as a model of human breast cancer metastasis to lung. We report that the procedure led to poor animal condition 7-8 weeks after injection, and produced lesions in the lungs visible at necropsy but we were unable identify individual cancer cells using immunohistochemistry. We conclude that if this method is to produce a model that can be used in drug experiments, improvements are required for cancer cell detection post mortem, such as by using of a fluorescently tagged human lung cancer cell line.

Curcumae Radix Extract Decreases Mammary Tumor-Derived Lung Metastasis via Suppression of C-C Chemokine Receptor Type 7 Expression.

Curcumae radix is the dry root of Curcuma longa L. (turmeric) that can be used either as a spice or traditional medicine. The aim of this study was to investigate the survival benefits and the anti-metastatic activity of curcumae radix extract (CRE) in MCF7 cells and in MMTV-PyMT transgenic mice—a mouse model of breast cancer metastasis. In vitro wound scratch assay revealed that CRE treatment inhibited cell motility and cell migration in a dose-dependent manner. To investigate the effect of CRE in breast cancer metastasis, MMTV-PyMT transgenic female virgin mice were used and randomly divided into two groups. For survival curve analysis, CRE was administered in a dose of 50 mg/kg to 8–20-week-old mice. Interestingly, CRE treatment significantly increased the median and prolonged survival of MMTV-PyMT mice. Furthermore, CRE treatment decreased tumor burden and inhibited cell proliferation in primary breast tumor, and also suppressed mammary tumor-derived lung metastasis. The size of the lung metastases substantially decreased in the CRE-treated group compared with the ones in the control group. Curcumae radix extract showed anti-metastatic activity through regulating the expression of metastasis markers including C-C Chemokine Receptor Type 7, Matrix Metalloproteinase 9 and the proto-oncogenes c-fos and c-jun. We demonstrated that these metastatic regulators were decreased when CCR7 expression was suppressed in MCF7 cells transfected with CCR7 siRNA. The results of this study show that curcumae radix exerts antitumor and anti-metastatic activities, and we suggest that curcumae radix might be a potential supplement for the treatment and prevention of breast cancer metastasis.

Does the mouse tail vein injection method provide a good model of lung cancer?

Lung cancer drug development requires screening in animal models. We aimed to develop orthotopic models of human non-small lung cancer using A549 and H3122 cells delivered by tail vein injection. This procedure has been used previously for a mouse lung cancer (Lewis lung carcinoma) and as a model of human breast cancer metastasis to lung. We report that the procedure led to poor animal condition 7-8 weeks after injection, and produced lesions in the lungs visible at necropsy but we were unable identify individual cancer cells using immunohistochemistry. We conclude that if this method is to produce a model that can be used in drug experiments, improvements are required for cancer cell detection post mortem, such as by using of a fluorescently tagged human lung cancer cell line.

Abstract P1-05-01: Breast cancer cell-derived IL-1B drives metastasis and colonisation of the bone microenvironment

Abstract Background: Breast cancer bone metastases are incurable and new therapeutic targets needed. After homing and colonising bone, cancer cells remain dormant, until signals from the microenvironment stimulate their proliferation to form overt metastases. We have recently identified interleukin-1B (IL-1B) as a potential marker for predicting breast cancer patients at increased risk of skeletal relapse and established a role for IL-1 signalling in tumour dormancy in bone. Here we present novel data to support that tumour cell-derived IL-1B plays major roles in breast cancer metastasis and growth in bone. Methods: Tumour/stromal IL-B and IL-1R1 expression was assessed in samples from the AZURE study following immunohistochemical staining. A humanised mouse model of MDA-MB-231 bone metastasis was used to assess effects of the IL-1R antagonist, Anakinra, on tumour growth and spontaneous metastasis. Effects of tumour cell-derived IL-1B on parameters associated with epithelial to mesenchymal transition and metastasis were measured by ELISA, QPCR, Western blot, transwell and scratch assays in MDA-MB-231, MCF7 and T47D cells transfected with IL-1B/control. Homing of breast cancer cells to bone was monitored in BALB/c nude mice intra venously injected with IL-1B overexpressing/control cells. To assess anti-tumour effects of IL-1 inhibition combined with standard of care, BALB/c or C57BL/6 mice were injected with 4T1 or E0771 cells (intra cardiac or intra ductal) 7-days prior to administration of Anakinra, doxorubicin, zoledronic acid (ZA) or placebo, alone or in combination for 14 days. Results: In tissue samples from &amp;gt;1300 patients with stage II/III breast cancer, active IL-1B in tumour cells correlated with relapse in bone (hazard ratio 1.85; 95% CI 1.05-3.26; P=0.02) and other sites (hazard ratio 2.09; 95% CI 1.26-3.48; P=0.0016). In a model of spontaneous human breast cancer metastasis to human bone, Anakinra significantly reduced metastasis to bone (from 80% in control animals to, 20% in Anakinra treated mice) and reduced the number of tumour cells shed into the circulation. Genetic manipulation of breast cancer cells to overexpress IL-1B demonstrated that exogenous production of IL-1B promoted EMT (decreased E-Cadherin, N-Cadherin and G-Catenin), invasion, migration and organ-specific homing in ER-ve (MDA-MB-231) and ER+ve (T47D and MCF7) cells in vitro and in vivo. Contact between tumour cells and osteoblasts or bone marrow cells increased IL-1B secretion from all three cell types. Exposure of tumour cells to IL-1B in the absence of bone cells did not stimulate tumour cell proliferation. Instead, elevated concentrations of IL-1B caused expansion of the bone metastatic niche (increased osteoblasts and blood vessels) that in turn stimulated tumour proliferation. Adding Anakinra to chemotherapy and ZA to mice injected with E0771 cells completely prevented bone metastasis formation and reduced extra-skeletal metastasis by 33%. In mice injected with 4T1 cells, the triple combination therapy reduced bone metastasis by 50% and extra-skeletal metastases by 65%. Conclusion: Our novel data demonstrate that IL-1B/IL-1R1 signalling plays an important role in breast cancer metastasis to bone. Pharmacological inhibition of IL-1B has potential as a novel treatment. Citation Format: Ottewell PD, Lefley DV, Freeman K, Gregory WM, Hanby AM, Spicer-Hadlington AR, Liu X, Bradbury SM, Hambley L, Allocca G, Cookson V, Kruithof de-Julio M, Coleman RE, Brown JE, Holen I, Tulotta C. Breast cancer cell-derived IL-1B drives metastasis and colonisation of the bone microenvironment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-01.

Abstract PD9-10: Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis

Abstract Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Cancer associated fibroblasts (CAFs) are the majority of stroma in BC and critical players in BC malignancy. For example, CAFs are the main source of SDF-1, a prominent chemokine in the tumor microenvironment (TME) that also imparts stem cell-like characteristics to BC cells. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. Stem-like CTCs and clusters have a greater propensity to establish metastasis. We recently identified circulating CAFs (cCAFs) in blood from patients with BC and in spontaneous, syngeneic, and xenograft mouse models of BC. cCAFs not only circulate individually, but are also found in clusters with CTCs. In this study, we examine the role of CAFs in promoting egress of stem-like CTCs (cCSCs), determine the capacity of stem-like CTCs to cluster with CAFs, and evaluate the involvement of CTC/cCAF clustering in augmenting BC metastasis. Methods: Our model employs NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines: the non-metastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis, and we used BALB/c mice injected with syngeneic 4T1 or 67nR cells to evaluate cCAFs, CTCs, and cluster egress in preclinical models. Mice were sacrificed at specific time points, and cardiac blood was collected. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were stained for IF and cCAFs, CTCs, cCSCs, and clusters were enumerated. Tumors from CAF co-injected mice were evaluated for their stem cell-like phenotype and re-implanted in mice to evaluate tumorigenicity and metastasis. Results: In spontaneous, syngeneic, and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs co-clusters appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. Co-inoculation of CAFs with BC cells resulted in a significant increase in tumor progression, metastasis, and in a substantially higher number of both individual cells and clusters in circulation. Dissociated tumor cells from CAF co-injected tumors had a higher proportion of CD44+stem cell-like cells (CSCs), enhanced ALDH-1 expression, and enhanced mammosphere formation. CD44+ CSCs, individually and in clusters, are found early on in the circulation of mice injected with dissociated tumor cells from CAF co-injected tumors. Upon re-implantation of CAF co-injected dissociated tumor cells without CAFs, dissociated tumor cells showed enhanced tumorigenicity and malignancy. Conclusion: CAFs are highly motile and cCAFs precede CTCs into circulation and can do so independently of tumor cells. CAFs sustain egress of tumor cells by augmenting malignancy and stemness of BC cells. cCAF clusters with the highly metastatic stem cell-like subset of CTCs bolster metastatic colonization. Targeting primary CAF function and/or cCAF/cCSC co-clusters may provide novel avenues to abrogate BC metastasis. Citation Format: Sharma U, Miller P, Medina Saenz K, Picon-Ruiz M, Morata-Tarifa C, Spartz A, Troness B, Park DN, Seagroves TN, Slingerland JM, Lippman ME, El-Ashry D. Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-10.

Early and late endothelial response in breast cancer metastasis in mice: simultaneous quantification of endothelial biomarkers using a mass spectrometry-based method.

ABSTRACTThe endothelium plays an important role in cancer metastasis, but the mechanisms involved are still not clear. In the present work, we characterised the changes in endothelial function at early and late stages of breast cancer progression in an orthotopic model of murine mammary carcinoma (4T1 cells). Endothelial function was analysed based on simultaneous microflow liquid chromatography–tandem mass spectrometry using multiple reaction monitoring (microLC/MS-MRM) quantification of 12 endothelium-related biomarkers, including those reflecting glycocalyx disruption – syndecan-1 (SDC-1), endocan (ESM-1); endothelial inflammation – vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin (E-sel); endothelial permeability – fms-like tyrosine kinase 1 (FLT-1), angiopoietin 2 (Angpt-2); and haemostasis – von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), as well as those that are pathophysiologically linked to endothelial function – adrenomedullin (ADM) and adiponectin (ADN). The early phase of metastasis in mouse plasma was associated with glycocalyx disruption (increased SDC-1 and ESM-1), endothelial inflammation [increased soluble VCAM-1 (sVCAM-1)] and increased vascular permeability (Angpt-2). During the late phase of metastasis, additional alterations in haemostasis (increased PAI-1 and vWF), as well as a rise in ADM and substantial fall in ADN concentration, were observed. In conclusion, in a murine model of breast cancer metastasis, we identified glycocalyx disruption, endothelial inflammation and increased endothelial permeability as important events in early metastasis, while the late phase of metastasis was additionally characterised by alterations in haemostasis.

Combining Desmopressin and Docetaxel for the Treatment of Castration-Resistant Prostate Cancer in an Orthotopic Model.

Desmopressin is a synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in breast cancer models. Docetaxel is a chemotherapy agent for castrate-resistant prostate cancer (CRPC). In this study, the ability of CRPC cells to grow and develop in vivo tumors in an animal model was evaluated, in order to investigate the anti-tumor effect of desmopressin in combination with docetaxel. The CRPC cell line PC3 was used for orthotopic inoculation in male athymic nude mice. The mice were randomly assigned to one of the four treatment groups: Control, docetaxel, desmopressin or combination therapy. Following the last treatment, tumors were excised and measured. Blood samples were processed for CTC analysis. Docetaxel treatment resulted in a significant reduction in tumor volume compared to control. The combination therapy resulted in even more significant reduction (31.2%) in tumor volume. There was a complete absence of CTCs in the combination group. Our pilot study demonstrated an enhanced efficacy of docetaxel-based therapy in combination with desmopressin.

Core needle biopsy promotes lung metastasis of breast cancer: An experimental study.

Core needle biopsy (CNB) may be used to diagnose early-stage breast cancer, but it may increase the risk of distant metastasis of tumor cells. The aim of the present study was to explore the effect of CNB on the distant metastasis of breast cancer. A total of 30 BALB/c mice were divided into two groups, namely biopsy and non-biopsy groups. The biopsy-related lung metastasis model (biopsy group) was established by the inoculation in the mammary fat pad of the mouse breast cancer cell line 4T1 combined with CNB. Flow cytometry, quantitative polymerase chain reaction analysis, morphological analysis, as well as other techniques, were used to evaluate the biological behavior of the tumors in the mouse model. A stable and reliable lung metastasis model of breast cancer was successfully established. The number of metastatic lung nodules in the biopsy group was significantly higher compared with that in the non-biopsy group (P<0.05). Compared with the non-biopsy group, the mRNA expression of transforming growth factor (TGF)-β1, SOX4 and Ezh2 in the biopsy group was significantly upregulated (P<0.05) and the number of natural killer (NK) cells detected by flow cytometry was increased, but the difference was not statistically significant (P>0.05). Therefore, CNB was found to promote the lung metastasis of breast cancer, and the underlying mechanism may be associated with epithelial-to-mesenchymal transition (EMT) mediated by the TGF-β1 signaling pathway.