Murine Model Of Metastatic Breast Cancer Research Articles

Evaluation of the immunomodulatory activity of thalidomide on tumor-associated macrophages in the 4T1 murine metastatic breast cancer model

ABSTRACT The present work evaluated the immunomodulatory effect of thalidomide (Thal) at different doses on tumor-associated macrophages (TAMs) using a mouse model of human breast cancer. Mice were inoculated with 4T1 cells in the left flank and treated with Thal once a day at concentrations of 50, 100, and 150mg/kg body weight from the 5th day until the 28th day of tumor inoculation. The tumors were sized, proliferation index and TAMs count were evaluated in primary tumors and metastatic lungs. In addition, the metastasis rate was evaluated in the lungs. Thal at 150mg/kg significantly decreased tumor growth, proliferation index, and TAMs infiltration in primary tumors. Conversely, a higher number of TAMs and lower proliferation index were observed in metastatic lungs in mice treated with 150mg/kg of Thal. Furthermore, Thal at 150mg/kg significantly decreased the metastatic nodules in the lungs. Our findings demonstrated that Thal treatment considerably decreased the primary tumor and lung metastasis in mice associated with different TAM infiltration effects in these sites.

Nanoscale Coordination Polymers for Combined Chemotherapy and Photodynamic Therapy of Metastatic Cancer.

Combination therapy enhances anticancer efficacy through synergistic effects of different drugs/modalities and can potentially address the challenges in the treatment of metastatic diseases. Here we report the design of carb/pyro nanoscale coordination polymer nanoparticles that carry carboplatin (carb) in the core and the photosensitizer pyrolipid (pyro) on the shell for the treatment of metastatic triple negative breast cancer. Upon light irradiation, carb/pyro generated reactive oxygen species to cause severe cell apoptosis and early calreticulin exposure. Upon intravenous injection and local light irradiation, carb/pyro significantly regressed tumor growth in the 4T1 murine metastatic breast cancer model. When combined with an anti-CD47 antibody, carb/pyro with light irradiation completely eradicated primary and metastatic 4T1 tumors in 50% mice. The anticancer efficacy of carb/pyro was also demonstrated in the CT26 murine colorectal cancer model.

A bilateral tumor model identifies transcriptional programs associated with patient response to immune checkpoint blockade

Immune checkpoint blockade (ICB) is efficacious in many diverse cancer types, but not all patients respond. It is important to understand the mechanisms driving resistance to these treatments and to identify predictive biomarkers of response to provide best treatment options for all patients. Here we introduce a resection and response-assessment approach for studying the tumor microenvironment before or shortly after treatment initiation to identify predictive biomarkers differentiating responders from nonresponders. Our approach builds on a bilateral tumor implantation technique in a murine metastatic breast cancer model (E0771) coupled with anti-PD-1 therapy. Using our model, we show that tumors from mice responding to ICB therapy had significantly higher CD8+ T cells and fewer Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) at early time points following therapy initiation. RNA sequencing on the intratumoral CD8+ T cells identified the presence of T cell exhaustion pathways in nonresponding tumors and T cell activation in responding tumors. Strikingly, we showed that our derived response and resistance signatures significantly segregate patients by survival and associate with patient response to ICB. Furthermore, we identified decreased expression of CXCR3 in nonresponding mice and showed that tumors grown in Cxcr3-/- mice had an elevated resistance rate to anti-PD-1 treatment. Our findings suggest that the resection and response tumor model can be used to identify response and resistance biomarkers to ICB therapy and guide the use of combination therapy to further boost the antitumor efficacy of ICB.

Visualisation of sentinel lymph node with indium-based near infrared emitting Quantum Dots in a murine metastatic breast cancer model.

Due to its non-invasiveness, high temporal resolution and lower cost, fluorescence imaging is an interesting alternative to the current method (blue dye and radiocolloid) of sentinel lymph node (SLN) mapping in breast cancer. Near-infrared (NIR) emitting cadmium-based Quantum Dots (QDs) could be used for this purpose; however, their wide application is limited because of the toxicity of heavy metals composing the core. Our recent work demonstrated that indium-based QDs exhibit a weak acute local toxicity in vivo compared to their cadmium-based counterparts. In the present study we confirmed the weak toxicity of CuInS2/ZnS QDs in different in vitro models. Further in vivo studies in healthy mice showed that In-based QDs could be visualised in SLN in a few minutes after administration with a progressive increase in fluorescence until 8 h. The quantity of indium was assessed in selected organs and tissues by inductively coupled plasma – mass spectroscopy (ICP-MS) as a function of post-injection time. QD levels decrease rapidly at the injection point in the first hours after administration with a parallel increase in the lymph nodes and to a lesser extent in the liver and spleen. In addition, we observed that 3.5% of the injected indium dose was excreted in faeces in the first 4 days, with only trace quantities in the urine. Metastatic spread to the lymph nodes may hamper its visualisation. Therefore, we further performed non-invasive fluorescence measurement of QDs in SLN in tumour-bearing mice. Metastatic status was assessed by immunohistology and molecular techniques and revealed the utmost metastatic invasion of 36% of SLN. Fluorescence signal was the same irrespective of SLN status. Thus, near-infrared emitting cadmium-free QDs could be an excellent SLN tracer.

Abstract 1417: Role of the matricellular protein SPARC in breast tumor growth and metastatic dissemination

Abstract Breast cancer is the leading cause of death on women worldwide. Whereas surgical resection and adjuvant therapy can cure well-confined primary tumors, metastatic disease is largely incurable because of its systemic nature and the resistance of disseminated tumor cells to existing therapeutic agents. SPARC (Secreted Protein Acidic and Rich in Cystein) is a matricellular protein whose normal expression is associated with remodeling tissues. In this context SPARC was described as involved in cell cycle and proliferation, invasion, adhesion, migration, angiogenesis and apoptosis. Expression array technology among other approaches identified SPARC as marker of poor prognosis, very often associated with most aggressive tumors in the vast majority of human cancer types. However, there is confusing data regarding the role of this protein during the development and progression of breast tumors. In order to achieve a better understanding of the role of this protein during breast cancer progression, we used the murine metastatic breast cancer model, 4T1. We found that silencing SPARC in these epithelial cells, after transduction with a lentiviral vector carrying a siRNA against murine SPARC, results in the completely loss of their metastatic capability (p<0.001) and inhibits the in vivo growth capability (p<0.001) in an immunocompetent murine model. This was consistently found in three different siRNA-expressing 4T1-derived cell clones. In order to identify genes involved in this SPARC-induced behavior we designed two microarray studies to compare gene expression in the c18-4T1 SPARC-deficient clone and the control SCR-4T1 cells. The first array was aimed to compare gene expression of cells grown in vitro (in vitro array) while the second array was carried out using orthotopic tumors and their derived lung metastasis (in vivo array). When analyzing the in vitro array, the comparative ontological analysis indicated that SPARC knockdown alters mainly the transcription of cell cycle and extracellular matrix response genes. In agreement, functional assays confirmed that the downregulation of SPARC in tumor cells enhances the G1/S checkpoint. In the in vivo array, the alteration of the transcription of immune response genes became evident, indicating a strong contribution of SPARC to the tumor implantation response. Moreover, the changes in the extracellular matrix and immune response gene expression were evident both in the metastasis and in the primary tumor. In addition, we were able to identify a “minimal SPARC-dependent metastasis signature,” found in early SPARC-expressing tumors but not in SPARC-deficient ones, including 22 genes with expression levels similar to those found in their derived metastasis. Validation of this signature by meta-analysis of previously published human tumor signatures might serve to further test the relevance of SPARC in breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1417. doi:1538-7445.AM2012-1417

Abstract 1456: Prostaglandin E2 receptor EP1 as a metastasis suppressor in breast cancer

Abstract The cyclooxygenase-2 enzyme (COX-2) is highly expressed in breast cancer and contributes to the metastatic process. Inhibition of COX-2 can decrease breast tumor growth and metastasis. Prostaglandin E2 (PGE2), the chief COX-2 product in tumors, has been identified as a predominant protumorigenic prostanoid. PGE2 mediates biological effects by binding to each of four EP receptors (EP1-4). EP1 is coupled to calcium mobilization and PKC; whereas, EP2 and EP4 activate PKA/cAMP, PI3K and ERK pathways. EP3 generally inhibits cAMP levels. EP4, expressed on malignant breast cells, promotes metastasis, but a role for EP1 in metastasis has not been investigated. Using a combination of immunohistochemistry, pharmacologic and genetic approaches, we studied EP1 expression in human breast tissue and function in a murine model of breast cancer metastasis. Our published studies indicate that EP1 was detected by immunohistochemistry in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas. Overall survival for women with tumors that were negative for nuclear EP1 was significantly worse than for women with EP1 expression. Additionally, pharmacologic antagonism and reduction of EP1 expression by shRNA increased metastatic capacity in our murine model of metastatic breast cancer. These data suggested that EP1 may function as a metastasis suppressor. We now report that murine mammary tumor cell lines 410, 410.4 and 66.1 have decreased mRNA levels of EP1 compared to normal murine mammary epithelial cell line EpH4. The EP1 receptor is normally coupled to calcium mobilization. Even though EP1 expression is decreased in mammary tumor cell lines, preliminary studies suggest the existence of functional EP1 receptor as PGE2 is capable of inducing calcium mobilization in 410.4 cells. In addition, treatment of 410 and 66.1 cells with 5-azacytidine is capable of increasing EP1 mRNA levels which may identify a clinically relevant strategy to increase expression of this protective receptor. We have also identified the presence of a variant EP1 (EP1v) transcript in this murine metastatic breast cancer model. EP1v has been identified previously in murine mast cell line MC/9 and in rat uterus; however, this is the first report that EP1v has been identified in malignant cells. Like EP1, mRNA expression of EP1v is also decreased in 410, 410.4 and 66.1 cell lines compared to EpH4 cells. Our published studies show that EP1 acts to suppress metastasis and we are currently exploring the contribution of EP1v to this mechanism. These findings suggest that EP1 has the potential to be a new therapeutic target in reducing breast cancer metastasis and increasing overall cancer survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1456. doi:10.1158/1538-7445.AM2011-1456

Identification of Metastasis-Related Proteins and Their Clinical Relevance to Triple-Negative Human Breast Cancer

Currently, there are no definite biomarkers of triple-negative breast cancer. The study aims to identify the metastasis-associated proteins of triple-negative breast tumors. A murine metastatic breast cancer model has been established by using TA2 mice. Parallel proteomic analyses were done on a murine metastatic breast cancer model and its primary breast cancer using two-dimensional gel electrophoresis. The differentially expressed proteins were detected in TA2 mice developing spontaneous breast cancer and lung metastasis. Furthermore, their expression were detected in human breast cancer with or without metastasis, and their prediction values were assessed in a second set of samples. Nineteen of 36 differentially expressed proteins were identified by peptide mass fingerprinting using matrix-assisted laser-desorption ionization-time of flight-mass spectrometry. These proteins were also validated in mouse tumor tissues by immunohistochemical staining. Actin, 14-3-3, vimentin, HSP70, CK18, and moesin were up-regulated in the metastatic tumors, whereas HSP90 and tubulin were absent or down-regulated. Furthermore, 61 patients with triple-negative breast cancer and 39 patients with estrogen receptor-positive/progesterone receptor-positive breast cancer were selected for exploring the clinical relevance of these identified proteins to human breast cancer metastasis. Expression of 14-3-3 and HSP70 was significantly correlated with metastasis of human triple-negative breast cancer. Moreover, the validation study in the second set confirmed that 14-3-3, HSP70, and their combination had high sensitivities and specificities in predicting metastatic potential of triple-negative breast cancer. These tumor metastasis-associated proteins validated may be useful as biomarkers and targets for diagnosis and treatment of human triple-negative breast cancer.