Mammary Pad Research Articles

Curcumin Administered in Combination with Glu-GNPs Induces Radiosensitivity in Transplanted Tumor MDA-MB-231-luc Cells in Nude Mice.

Curcumin is a type of plant polyphenol extracted from Curcuma longa L. rhizome, which demonstrates antitumor activity in breast cancer cells in vitro. To investigate the combined effect and possible mechanism of curcumin and glucose-gold nanoparticles (Glu-GNPs), the radiosensitivity of breast carcinoma xenografts was assessed in nude mice. MDA-MB-231 cells labeled with firefly luciferase were inoculated into the mammary fatty pads of nude mice to establish a transplantation tumor model of human breast cancer. The tumor-bearing mice were treated with different drugs (curcumin, Glu-GNPs, and cisplatin) for 3 weeks prior to radiotherapy. The body weights and tumor volumes of the mice were measured in regular intervals. Tumor bioluminescence intensity was determined in real-time using an in vivo bioluminescence imaging system to monitor tumor growth. Transplanted tumor tissue samples were taken for hematoxylin and eosin (HE) staining, and the expression of VEGF, HSP90, HIF-1α, and MMP9 was evaluated via reverse transcription-quantitative PCR or immunohistochemistry. The results revealed that the breast tumor-bearing nude mouse model was successfully established, as evidenced by a stable expression of luciferase. Curcumin inhibited the growth of tumors without causing significant weight loss in mice. Furthermore, additive inhibition was demonstrated when curcumin was administered in combination with Glu-GNPs and irradiation. Tumor bioluminescence intensity was decreased in the model group following curcumin, Glu-GNPs, and irradiation treatment. HE staining demonstrated that transplanted tumors were malignant, with necrotic tissue exhibited centrally. It was concluded that curcumin administered in combination with Glu-GNPs and X-ray irradiation could reduce the protein expression of VEGF, HSP90, HIF-1α, and MMP9 in tumor tissue when compared with the model group. Curcumin and Glu-GNPs administered with X-ray irradiation significantly inhibited tumor growth and induced radiosensitivity, which may be associated with the inhibition of angiogenesis in tumor tissue.

Resetting the tumor microenvironment to favor anti-tumor immunity after local ablation.

2561 Background: Percutaneous tumor ablation is a non-surgical method of tumor destruction that leaves necrotic tumor debris in situ. Tumor associated antigens released after ablation have the potential to initiate a systemic anti-tumor immune response, however the hostile tumor microenvironment hinders antigen presentation and T cell activity. We hypothesized that resetting the tumor microenvironment with oral sodium bicarbonate to decrease tumor acidity and low-dose cyclophosphamide to deplete pro-tumor immune cells would improve the ability of ablation to initiate anti-tumor immunity. Methods: Tumor growth, overall survival, and metastatic burden was assessed in orthotopic tumor models of triple-negative breast cancer (67NR, 4T1, and E0771). Tumor ablation was performed on palpable tumors using percutaneous ethanol injection (PEI) with 6% ethylcellulose to improve retention in the tumor. Surgical excision was used as a negative control to test the role of in situ tumor debris. Before ablation mice were placed on 200 mM of sodium bicarbonate (SB) in their drinking water and received a single intraperitoneal injection of 200 mg/kg of cyclophosphamide (CP). Mice surviving to 60 days after tumor implant without a primary tumor or signs of metastases were considered "cured" and re-challenged with 50e5 tumor cells in the contralateral mammary pad. T cell dependance was assessed with in vivo CD8 depletions. Results: The combination of PEI+SB+CP produced a potent anti-tumor response, curing a majority of mice (5/7 of E0771, 8/12 of 67NR, 7/12 of 4T1). No mice were cured using PEI alone, SB alone, CP alone, or any combination of two therapies (0/51 of E0771, 0/73 of 67NR, 0/75 of 4T1,). Re-challenge tumor growth was hindered in mice cured with PEI+SB+CP. Mice receiving PEI+SB+CP had significantly less metastases and lived longer than mice receiving surgical excision alone or surgical excision with SB+CP. Additionally the anti-metastatic response of PEI+SB+CP was undone when CD8+ T cells were depleted. Conclusions: Here the anti-tumor response of local ablation produced by PEI was enhanced by priming the tumor with low-dose CP and oral SB in metastatic breast cancer. These results suggest that tumor ablation with CP and SB can create a T cell dependent, personalized immune response to a tumor using only low-cost, easily accessible supplies, and the host’s own tumor.

Melatonin decreases metastasis, primary tumor growth and angiogenesis in a mice model of breast cancer.

The goal of this study was to mechanistically analyze the effects of pre-treatment or post-treatment melatonin on the metastatic spread in a mice model. Consequently, the effects on the tumor growth, angiogenesis and metastasis were evaluated with immunohistochemical and western blot analysis. 8-10 weeks-old female BALB/c mice (n = 60, 10/group) were used. Liver metastatic cells (4TLM) from 4T1 murine breast carcinoma were previously isolated. Melatonin was administrated either before or after the injection of 4TLM cells into the mammary pad. Tumor and vehicle (%6 ethanol) injections were given to vehicle groups. Tumor group consisted of the mice injected with only 4TLM cells injected to tumor group and no intervention to control group. Necropsies were performed 27 days after injection of 4TLM. Primary tumors and metastatic tissues were removed. Furthermore, changes in lung and liver metastasis and primary tumor growth and angiogenesis were evaluated. In our study neutrophil levels were noted to be increased in peripheral blood of the tumor-bearing mice. Melatonin exerted inhibitory effects on the 4TLM-induced leukocytosis. Melatonin significantly decreased lung and liver metastasis, primary tumor growth and angiogenesis. The results demonstrated that melatonin might have a therapeutic role through reducing systemic inflammatory responses, metastasis, tumor growth and angiogenesis.

Abstract PS3-29: Pixel-level tissue classification from ultrasound images of breast cancer and direct comparison to matched histological measurements

Abstract Introduction: The use of noninvasive ultrasound (US) for quantitative tissue characterization has been an exciting research prospect for several decades now. Herein the challenge is to find hidden patterns in the US data to reveal more information about tissue function and pathology that cannot be seen in the more conventional US images. A new pixel-level analysis technique has been developed by our group for tissue classification. Termed H-scan US (H stands for hue or Hermite), this imaging approach links a special class of nth-ordered Gaussian-weighted Hermite functions (GHn) to the physics of US scattering and reflection from different tissue structures. The sensitivity of in vivo H-scan US imaging to subtle changes in scatterer size is a question that has not been fully answered. To that end, the purpose of this study was to compare local H-scan US image intensity to direct (co-registered) histological measures made at the cellular level. Methodology Female nude athymic mice were implanted into mammary fatty pad with breast cancer cells (N = 8). Implanted tumors were allowed to grow for about four weeks before study enrollment. Image data was acquired using a programmable US scanner (Vantage 256, Verasonics Inc) equipped with a 256-element L22-8v CMUT linear array transducer (Kolo Medical). Plane wave imaging with 5 angles was performed at a center frequency of 15 MHz. To generate the H-scan US image, three parallel convolution filters (GH2, GH6, and GH10) were applied to the radiofrequency (RF) data sequences to measure the relative strength of the received signals. After envelope detection, the relative strength of the filter outputs is color coded whereby the lower frequency (GH2 = 9 MHz) backscattered US signal components are assigned to the R channel, moderate frequency (GH6 = 15 MHz) signals are assigned to the G channel, and the higher frequency (GH10 = 21 MHz) signals to the B channel. After performing H-scan US, the imaging cross-section was marked, and the tumors were excised and sliced along the same plane for histologic processing. After nuclear staining, tissue sections were scanned and digitized using confocal microscopy and fluorescent filters for DAPI. Automated segmentation of each cancer cell nucleus in the histologic sections was performed using an active contour technique. US images were interpolated to the same number of pixels as the histology image and spatial alignment between the two was performed. Lastly, nucleus size and density from histologic sections was compared to local H-scan US image features. Results: Custom software was developed to compare local US image features at the pixel level to that of co-registered histologic images and measurements of nuclear size and density. In an attempt to properly match the histological and the H-scan US images, three different sized kernels of 0.16 ✗ 0.16 mm, 0.32 ✗ 0.32 mm or 0.5 ✗ 0.5 mm were used to partition both the H-scan US and histology images into 1888, 448 or 62 distinct region-of-interests (ROIs), respectively. Mean nuclear size and density measurements from the histology ROIs was compared to the mean H-scan US image intensity from the many spatially matched ROI locations. A statistically significant linear relationship was found between local H-scan US image intensity and nuclear size (R2 > 0.4, < 0.001) and density (R2 > 0.6, p < 0.001). Conclusions: Preliminary results from use of an animal model of breast cancer reveals that in vivo H-scan US images positively correlated with physical measures of nucleus size and density as quantified from co-registered histologic images. Citation Format: Mawia Khairalseed, Shreya Reddy, Jane Song, Girdhari Rijal, Kenneeth Hoyt. Pixel-level tissue classification from ultrasound images of breast cancer and direct comparison to matched histological measurements [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-29.

Mechanical Loading-Driven Tumor Suppression Is Mediated by Lrp5-Dependent and Independent Mechanisms.

Simple SummaryAdvanced breast cancer and prostate cancer metastasize to varying organs including the bone. We show here that mechanical loading to the knee suppresses tumor growth in the loaded bone and the non-loaded mammary pad. Although lipoprotein receptor-related protein 5 (Lrp5) in osteocytes is necessary to induce loading-driven bone formation, loading-driven tumor suppression is regulated by Lrp5-dependent and independent mechanisms. Lrp5 overexpression in osteocytes enhances tumor suppression, but without Lrp5 in osteocytes, mechanical loading elevates dopamine, chemerin, p53, and TNF-related apoptosis-inducing ligand (TRAIL) and reduces cholesterol and nexin. Their systemic changes contribute to inhibiting tumors without Lrp5. Osteoclast development is also inhibited by the load-driven regulation of chemerin and nexin.Bone is mechanosensitive and lipoprotein receptor-related protein 5 (Lrp5)-mediated Wnt signaling promotes loading-driven bone formation. While mechanical loading can suppress tumor growth, the question is whether Lrp5 mediates loading-driven tumor suppression. Herein, we examined the effect of Lrp5 using osteocyte-specific Lrp5 conditional knockout mice. All mice presented noticeable loading-driven tumor suppression in the loaded tibia and non-loaded mammary pad. The degree of suppression was more significant in wild-type than knockout mice. In all male and female mice, knee loading reduced cholesterol and elevated dopamine. It reduced tumor-promoting nexin, which was elevated by cholesterol and reduced by dopamine. By contrast, it elevated p53, TNF-related apoptosis-inducing ligand (TRAIL), and chemerin, and they were regulated reversely by dopamine and cholesterol. Notably, Lrp5 overexpression in osteocytes enhanced tumor suppression, and osteoclast development was inhibited by chemerin. Collectively, this study identified Lrp5-dependent and independent mechanisms for tumor suppression. Lrp5 in osteocytes contributed to the loaded bone, while the Lrp5-independent regulation of dopamine- and cholesterol-induced systemic suppression.

In Vivo Optical Metabolic Imaging of Long-Chain Fatty Acid Uptake in Orthotopic Models of Triple-Negative Breast Cancer.

Simple SummaryA dysregulated metabolism is a hallmark of cancer. Once understood, tumor metabolic reprogramming can lead to targetable vulnerabilities, spurring the development of novel treatment strategies. Beyond the common observation that tumors rely heavily on glucose, building evidence indicates that a subset of tumors use lipids to maintain their proliferative or metastatic phenotype. This study developed an intra-vital microscopy method to quantify lipid uptake in breast cancer murine models using a fluorescently labeled palmitate molecule, Bodipy FL c16. This work highlights optical imaging’s ability to both measure metabolic endpoints non-destructively and repeatedly, as well as inform small animal metabolic phenotyping beyond in vivo optical imaging of breast cancer alone.Targeting a tumor’s metabolic dependencies is a clinically actionable therapeutic approach; however, identifying subtypes of tumors likely to respond remains difficult. The use of lipids as a nutrient source is of particular importance, especially in breast cancer. Imaging techniques offer the opportunity to quantify nutrient use in preclinical tumor models to guide development of new drugs that restrict uptake or utilization of these nutrients. We describe a fast and dynamic approach to image fatty acid uptake in vivo and demonstrate its relevance to study both tumor metabolic reprogramming directly, as well as the effectiveness of drugs targeting lipid metabolism. Specifically, we developed a quantitative optical approach to spatially and longitudinally map the kinetics of long-chain fatty acid uptake in in vivo murine models of breast cancer using a fluorescently labeled palmitate molecule, Bodipy FL c16. We chose intra-vital microscopy of mammary tumor windows to validate our approach in two orthotopic breast cancer models: a MYC-overexpressing, transgenic, triple-negative breast cancer (TNBC) model and a murine model of the 4T1 family. Following injection, Bodipy FL c16 fluorescence increased and reached its maximum after approximately 30 min, with the signal remaining stable during the 30–80 min post-injection period. We used the fluorescence at 60 min (Bodipy60), the mid-point in the plateau region, as a summary parameter to quantify Bodipy FL c16 fluorescence in subsequent experiments. Using our imaging platform, we observed a two- to four-fold decrease in fatty acid uptake in response to the downregulation of the MYC oncogene, consistent with findings from in vitro metabolic assays. In contrast, our imaging studies report an increase in fatty acid uptake with tumor aggressiveness (6NR, 4T07, and 4T1), and uptake was significantly decreased after treatment with a fatty acid transport inhibitor, perphenazine, in both normal mammary pads and in the most aggressive 4T1 tumor model. Our approach fills an important gap between in vitro assays providing rich metabolic information at static time points and imaging approaches visualizing metabolism in whole organs at a reduced resolution.

Enhancing the in vitro and in vivo activity of itraconazole against breast cancer using miltefosine-modified lipid nanocapsules

Itraconazole (ITC), a well-tolerated antifungal drug, exerts multiple anticancer effects which justified its preclinical and clinical investigation as potential anti-cancer agent with reduced side effects. Enhancement of ITC anti-cancer efficacy would bring valuable benefits to patients. We propose herein lipid nanocapsules (LNCs) modified with a subtherapeutic dose of miltefosine (MFS) as a membrane bioactive amphiphilic additive (M-ITC-LNC) for the development of an ITC nanoformulation with enhanced anticancer activity compared with ITC solution (ITC-sol) and unmodified ITC-LNC. Both LNC formulations showed a relatively small size (43–46 nm) and high entrapment efficiency (>97%), though ITC release was more sustained by M-ITC-LNC. Cytotoxicity studies revealed significantly greater anticancer activity and selectivity of M-ITC-LNC for MCF-7 breast cancer cells compared with ITC-sol and ITC-LNC. This trend was substantiated by in vivo findings following a 14 day-treatment of murine mammary pad Ehrlich tumors. M-ITC-LNC showed the greatest enhancement of the ITC-induced tumor growth inhibition, proliferation, and necrosis. At the molecular level, the tumor content of Gli 1, caspase-3, and vascular endothelial growth factor verified superiority of M-ITC-LNC in enhancing the ITC antiangiogenic, apoptotic, and Hedgehog pathway inhibitory effects. Finally, histopathological and biochemical analysis indicated greater reduction of ITC systemic toxicity by M-ITC-LNC. Superior performance of M-ITC-LNC was attributed to the effect of MFS on the structural and release properties of LNC coupled with its distinct bioactivities. In conclusion, MFS-modified LNC provides a simple nanoplatform integrating the potentials of LNC and MFS for enhancing the chemotherapeutic efficacy of ITC and possibly other oncology drugs.

Effects of obesity on NK cells in a mouse model of postmenopausal breast cancer

Obesity is a widely spread disease and a crucial risk factor for malign disorders, including breast cancer of women in the postmenopause. Studies demonstrated that in case of obesity crucial natural killer (NK) cell functions like combating tumor cells are affected. This study aims to analyze NK cells and NK cell receptor expression of obese mice in a model for postmenopausal breast cancer. Therefore, female BALB/c mice were fed either a high fat or a standard diet. Thereafter, ovaries were ectomized and a syngeneic and orthotopical injection of 4T1-luc2 mouse mammary tumor cells into the mammary adipose tissue pad was performed. Obese mice showed increased body weights and visceral fat mass as well as increased levels of leptin and IL-6 in plasma. Moreover, compared to the lean littermates, tumor growth was increased and the NKp46-expression on circulating NK cells was decreased. Furthermore, the activating NK cell receptor NKG2D ligand (MULT1) expression was enhanced in adipose tissue of obese tumor bearing mice. The present study gives novel insights into gene expression of NK cell receptors in obesity and aims to promote possible links of the obesity-impaired NK cell physiology and the elevated breast cancer risk in obese women.

Abstract 3963: Physical confinement induces malignant transformation in mammary epithelial cells

Abstract The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics. Citation Format: Yen-Chun Lu. Physical confinement induces malignant transformation in mammary epithelial cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3963.

Abstract 3687: Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells

Abstract ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic surface antigen that is expressed on breast cancer, but not on normal postpartum tissues. We found ROR1 was a receptor for Wnt5a, and high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). More recently, ROR1 could activate RhoGTPases and enhance tumor growth in breast cancer (Zhang S., et al., PNAS, 116(4), 2019). Such effects of Wnt5a on breast cancer patient-derived-xenograft (PDX) tumors could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. However, the molecular mechanism(s) for how ROR1 contributes such functions remained elusive. We performed mass spectrometry-based proteomic analysis on anti-ROR1 immune precipitates from breast cancer PDX tumors and detected DOCK1 in addition to ROR1, implying that ROR1 associates with DOCK1. DOCK1 (Dedicator of cytokinesis 1, also known as DOCK180) that expressed in breast cancer PDXs and cell lines, is a cytoplasmic protein, and a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1. Moreover, DOCK1 consists SH3 domain, which allows it to bind characteristic motifs (-P-X-X-P-), which often are found in the proline rich domains (PRD) of other proteins. Immunoblot analysis confirmed that ROR1 associated with DOCK1 in breast cancer PDX tumors or MCF7 cells transfected to express ROR1 (MCF7-ROR1) in response to Wnt5a, leading to enhanced activation of Rac1. To explore functional role, we performed a 2-week colony-formation assay using MCF7-ROR1 cells, and found that treatment of Wnt5a could enhance number of colonies of MCF7-ROR1 cells and that such colony formation capability could be inhibited by reducing expression of DOCK1 with specific siRNA, but not by control siRNA, suggesting a dependency on DOCK1 for Wnt5a-enhanced MCF7-ROR1 cells colony formation capability. Moreover, such effects of Wnt5a to enhance colony formation ability of MCF7-ROR1 cells could be inhibit by cirmtuzumab. We also introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1 proline-rich-domain (PRD) at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P⇒A mutants, ROR1P(808)A was unable to recruit DOCK1 to ROR1, or activate Rac1 in response to Wnt5a. Next we examined the tumor-growth potential of MCF7-Ctrl, MCF7-ROR1, or MCF7-P808A cells. The cells (5 × 104 cells/inoculate in RPMI 1640 together with an equal volume of Matrigel) were injected subcutaneously into the second right mammary pad of 4-to 6-week-old Rag2−/−γc−/- mice pretreated with estrogen pellets (17β-estradiol), which were placed subcutaneously in the intrascapular region one week before the cells inoculation. We found that MCF7-ROR1 cells (n=6) had significantly higher tumor growth relative to MCF7 cells (n=8), which had the same tumor growth as that of MCF7-P808A cells (n=6). Moreover, MCF7-ROR1P808A tumors had less activation of Rac1 compared to MCF7-ROR1 cells expressing wild-type ROR1. This study reveals that the recruitment of DOCK1 to ROR1 may be critical for the capacity of Wnt5a to enhance breast cancer tumor growth. Citation Format: Md Kamrul Hasan, George F. Widhopf II, Suping Zhang, Sharon M. Lam, Barbara A. Parker, Thomas J. Kipps. Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3687.

XIAOPI formula inhibits the pre-metastatic niche formation in breast cancer via suppressing TAMs/CXCL1 signaling

BackgroundRecent findings suggested that premetastatic niche (PMN) is a prerequisite in mediating cancer metastasis. Previously we demonstrated that XIAOPI formula could inhibit breast cancer lung metastasis via inhibiting tumor associated macrophages (TAMs)-secreted CXCL1. Herein, we aimed to explore the effects of XIAOPI formula on preventing breast cancer PMN formation and its underlying molecular mechanisms.MethodsCXCL1 expression of TAMs was detected by qPCR and Western blotting assay. The influences of XIAOPI formula on the proliferation of TAMs and 4 T1 in the co-culture system were tested by CCK8 or EdU staining. Transwell experiment was applied to determine the effects of XIAOPI formula on the invasion ability of HSPCs and 4 T1. Breast cancer xenografts were built by inoculating 4 T1 cells into the mammary pads of Balb/c mice and lung metastasis was monitored by luciferase imaging. Immune fluorescence assay was used to test the epithelial-mesenchymal transition process and PMN formation in the lung tissues. The effects of XIAOPI formula on TAMs phenotype, hematopoietic stem/progenitor cells (HSPCs) and myeloid-derived suppressor cells (MDSCs) were determined by flow cytometry.ResultsIt was found that XIAOPI formula could inhibit the proliferation and polarization of M2 phenotype macrophages, and reduce CXCL1 expression in a dose-dependent manner. However, M1 phenotype macrophages were not significantly affected by XIAOPI formula. TAMs/CXCL1 signaling was subsequently found to stimulate the recruitment of c-Kit+/Sca-1+ HSPCs and their differentiation into CD11b+/Gr-1+ MDSCs, which were symbolic events accounting for PMN formation. Moreover, XIAOPI formula was effective in inhibiting HSPCs activation and suppressing the proliferation and metastasis of breast cancer cells 4 T1 induced by HSPCs and TAMs co-culture system, implying that XIAOPI was effective in preventing PMN formation in vitro. Breast cancer xenograft experiments further demonstrated that XIAOPI formula could inhibit breast cancer PMN formation and subsequent lung metastasis in vivo. The populations of HSPCs in the bone marrow and MDSCs in the lung tissues were all remarkably declined by XIAOPI formula treatment. However, the inhibitory effects of XIAOPI formula could be relieved by CXCL1 overexpression in the TAMs.ConclusionsTaken together, our study provided preclinical evidence supporting the application of XIAOPI formula in preventing breast cancer PMN formation, and highlighted TAMs/CXCL1 as a potential therapeutic strategy for PMN targeting therapy.4rAD-F1p2pRhmA-5xKKeeVVideo Graphical abstract

Abstract P1-03-07: Investigation of combination treatment an aromatase inhibitor and anti-inflammatory treatment in a model of oestrogen receptor positive breast cancer

Abstract Background: Previous analysis has revealed that high pre-treatment expression of an inflammatory gene expression signature and lymphocytic infiltration are correlated with poor response to neoadjuvant aromatase inhibitor (AI) treatment1. Upon treatment with AIs, inflammatory gene expression also increases1. These changes are also seen in vitro where an increase in chemokine secretion has been observed upon oestrogen deprivation, suggesting that treatment with anti-oestrogen therapy may modulate the immune response. In addition, non-steroidal anti-inflammatory drug (NSAID) intake is associated with a significantly decreased risk of disease recurrence and breast cancer related death. In this study we set out to investigate the immunological response to AI treatment alone and in combination with NSAID treatment in an animal model of estrogen receptor positive (ER+ve) breast cancer. Methods: A syngeneic immunocompetent murine ER+ breast tumour model was used in which SSM3 ER+ BrCa cells were injected into the mammary pad of wild-type 129SvEv mice. Once tumours reached a size of 49 mm2, mice were ovariectomised and treated with combinations of letrozole, celecoxib, aspirin and vehicle control. Tumours were measured for 25 days following treatment and tumours were analysed using flow cytometry and immunohistochemistry. Results: Treatment with letrozole alone and letrozole + celecoxib resulted in a significant reduction tumour size after 25 days of treatment (p<0.001). Examination of the immune cell infiltrate of these tumours using flow cytometry determined that combination letrozole and celecoxib resulted in decreased numbers of CD11b+ cells compared to vehicle treated tumours (p = 0.02), and to celecoxib only treated tumours (p = 0.008). Working within recommended guidelines for assessing stromal TILs in H&E sections in a larger cohort (n=51), we determined that the percentage of stromal TILs increased in all treatment groups compared to the pre-treatment biopsy, and that the combination treatment had lower levels of TILs compared to vehicle treated tumours (p = 0.02). Conclusions: We conclude that treatment of ER+ BrCa with letrozole induces an immune response in the tumour microenvironment which can be modulated by the addition of an NSAID. Our findings suggest that modulation of the immune response to endocrine therapy may have the potential to enhance response to treatment.

Novel Breast Cancer Brain Metastasis Patient-Derived Orthotopic Xenograft Model for Preclinical Studies.

The vast majority of mortality in breast cancer results from distant metastasis. Brain metastases occur in as many as 30% of patients with advanced breast cancer, and the 1-year survival rate of these patients is around 20%. Pre-clinical animal models that reliably reflect the biology of breast cancer brain metastasis are needed to develop and test new treatments for this deadly condition. The patient-derived xenograft (PDX) model maintains many features of a donor tumor, such as intra-tumor heterogeneity, and permits the testing of individualized treatments. However, the establishment of orthotopic PDXs of brain metastasis is procedurally difficult. We have developed a method for generating such PDXs with high tumor engraftment and growth rates. Here, we describe this method and identify variables that affect its outcomes. We also compare the brain-orthotopic PDXs with ectopic PDXs grown in mammary pads of mice, and show that the responsiveness of PDXs to chemotherapeutic reagents can be dramatically affected by the site that they are in.

Effects of Lidocaine and Src Inhibition on Metastasis in a Murine Model of Breast Cancer Surgery.

Breast cancer recurs in 20% of patients following intended curative resection. In vitro data indicates that amide local anaesthetics, including lidocaine, inhibit cancer cell metastasis by inhibiting the tyrosine kinase enzyme Src. In a murine breast cancer surgery model, systemic lidocaine reduces postoperative pulmonary metastases. We investigated whether the additional administration of bosutinib (a known Src inhibitor) influences lidocaine’s observed beneficial effect in this in vivo model. Female BALB/c mice (n = 95) were inoculated with 25,000 4T1 cells into the mammary fad pad and after 7 days the resulting tumours were excised under sevoflurane anaesthesia. Experimental animals were randomized to one of four treatments administered intravenously prior to excision: lidocaine, bosutinib, both lidocaine and bosutinib in combination, or saline. Animals were euthanized 14 days post-surgery and lung and liver metastatic colonies were evaluated. Post-mortem serum was analysed for MMP-2 and MMP-9, pro-metastatic enzymes whose expression is influenced by the Src pathway. Lidocaine reduced lung, but not liver metastatic colonies versus sevoflurane alone (p = 0.041), but bosutinib alone had no metastasis-inhibiting effect. When combined with lidocaine, bosutinib reversed the anti-metastatic effect observed with lidocaine on sevoflurane anaesthesia. Only lidocaine alone reduced MMP-2 versus sevoflurane (p = 0.044). Both bosutinib (p = 0.001) and bosutinib/lidocaine combined (p = 0.001) reduced MMP-9 versus sevoflurane, whereas lidocaine alone did not. In a murine surgical breast cancer model, the anti-metastatic effects of lidocaine under sevoflurane anaesthesia are abolished by the Src inhibitor bosutinib, and lidocaine reduces serum MMP-2. These results suggest that lidocaine may act, at least partly, via an inhibitory effect on MMP-2 expression to reduce pulmonary metastasis, but whether this is due to an effect on Src or via another pathway remains unclear.

Abstract 3259: heterodimeric IL-15 monotherapy results in complete regression of EO771 murine breast tumors through cDC1-lymphocyte interactions and induction of antitumor immunity

Abstract Introduction: IL-15 is a cytokine important for the maintenance, proliferation and activation of lymphocytes, including CD8+ T and natural killer (NK) cells. The native form of IL-15 is a heterodimer either embedded in the plasma membrane or released in a soluble form. Several preclinical cancer models have supported the anti-tumor activity of heterodimeric IL-15, which is presently in clinical trials. We have produced heterodimeric IL-15 (hetIL-15) and also fusions to the Fc fragments (hetIL-15FC) of human, macaque or mouse antibodies. We assessed the effects of hetIL-15 monotherapy after systemic and locoregional administration in the proximity of EO771 orthotopically implanted breast tumors. Experimental procedures: We evaluated the therapeutic efficacy of hetIL-15 and hetIL-15FC immunotherapy in the murine EO771 triple negative breast cancer orthotopic model using both syngeneic C57BL/6 mice and RAG-1 immunodeficient mice. We monitored the effects of hetIL-15 treatment on Tumor Infiltrating Lymphocytes (TILs), lymphoid organs and blood by Flow cytometry and Immunohistochemisty (IHC). Using Seahorse technology, we also evaluated the metabolic profile of the CD8+ T cells, reflecting their activation status. Results: Monotherapy with hetIL-15FC peritumorally in the area of mammary pad resulted in complete tumor regression in half of the treated mice. hetIL-15 treatment resulted in an increased survival accompanied with decreased or completely eradicated metastatic disease, in C57BL/6 mice. Significant tumor delay was also observed after peritoneal injection of hetIL-15 in C57BL/6 as well as in RAG-1 mice with hetIL-15FC, but complete tumor regression was not achieved. hetIL-15FC peritumoral administration reshaped the tumor microenvironment and caused an increased infiltration of NK cells and CD8+ T cells with effector phenotype. Both cytotoxic T cells and NK were activated and proliferating, as shown by Flow, including Granzyme B and Ki67 staining. hetIL-15FC treatment resulted in increased tumor infiltration of both CD8+ and CD4+ T memory cells and provided strong protection to these mice against subsequent re-challenges with EO771 tumor. Furthermore, peritumoral hetIL-15FC administration resulted in an increase of intratumoral conventional Dendritic cells (cDC1), suggesting a mechanism for the intratumoral attraction of lymphocytes. Conclusion: Monotherapy with hetIL-15FC locoregional administration resulted in complete regression of EO771 primary breast cancer tumors, activating both cytotoxic arms of the immune system, and providing persisting protective immunity. Mechanism of function of hetIL-15 is through cDC1 and lymphocyte interactions that lead to the reciprocal attraction of cDC1 and activated cytotoxic lymphocytes into the tumors and the generation of long-term anti-tumor immunity. Citation Format: Dimitris Stellas, Sevasti Karaliota, Vasiliki Stravokefalou, Bethany A. Nagy, Barbara K. Felber, George N. Pavlakis. heterodimeric IL-15 monotherapy results in complete regression of EO771 murine breast tumors through cDC1-lymphocyte interactions and induction of antitumor immunity [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 3259.

Physical confinement induces malignant transformation in mammary epithelial cells

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement – a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4–6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.

Endurance training slows breast tumor growth in mice by suppressing Treg cells recruitment to tumors

BackgroundAerobic exercise has been shown to slow tumor progression in rodents and humans, but the mechanisms behind this effect are still unclear. Here we show that aerobic exercise in the form of chronic endurance training suppresses tumor recruitment of FoxP3+ Treg cells thus enhancing antitumor immune efficiency.MethodsAdult wild-type and athymic BALB/c female mice were endurance-trained for 8 weeks. Circulating leukocytes as well as muscle and liver mtDNA copy number were compared to aged-matched concurrent sedentary controls to establish systemic effects. 4 T1 murine mammary tumor cells were injected subcutaneously to the 4th mammary pad at the end of the training period. Tumor growth and survival rates were compared, together with antitumor immune response.ResultsExercised wild-type had 17% slower growth rate, 24% longer survival, and 2-fold tumor-CD+ 8/FoxP3+ ratio than sedentary controls. Exercised athymic BALB/c females showed no difference in tumor growth or survival rates when compared to sedentary controls.ConclusionsCytotoxic T cells are a significant factor in endurance exercise-induced suppression of tumor growth. Endurance exercise enhances antitumor immune efficacy by increasing intratumoral CD8+/FoxP3+ ratio.

Detection of Volatile Organic Compounds (VOCs) in Urine via Gas Chromatography-Mass Spectrometry QTOF to Differentiate Between Localized and Metastatic Models of Breast Cancer

Breast cancer is the most common cancer detected in women and current screening methods for the disease are not sensitive. Volatile organic compounds (VOCs) include endogenous metabolites that provide information about health and disease which might be useful to develop a better screening method for breast cancer. The goal of this study was to classify mice with and without tumors and compare tumors localized to the mammary pad and tumor cells injected into the iliac artery by differences in VOCs in urine. After 4T1.2 tumor cells were injected into BALB/c mice either in the mammary pad or into the iliac artery, urine was collected, VOCs from urine headspace were concentrated by solid phase microextraction and results were analyzed by gas chromatography-mass spectrometry quadrupole time-of-flight. Multivariate and univariate statistical analyses were employed to find potential biomarkers for breast cancer and metastatic breast cancer in mice models. A set of six VOCs classified mice with and without tumors with an area under the receiver operator characteristic (ROC AUC) of 0.98 (95% confidence interval [0.85, 1.00]) via five-fold cross validation. Classification of mice with tumors in the mammary pad and iliac artery was executed utilizing a different set of six VOCs, with a ROC AUC of 0.96 (95% confidence interval [0.75, 1.00]).

Oncologic Safety of Fat Grafting for Autologous Breast Reconstruction in an Animal Model of Residual Breast Cancer.

Clinical outcomes suggest that postoncologic reconstruction with fat grafting yields cumulative incidence curves of recurrence comparable to those of other breast reconstruction procedures; however, results from experimental research studies suggest that adipose stem cells can stimulate cancer growth. In this study, a novel animal model of residual cancer was developed in mouse mammary pads to test whether lipofilling impacts the probability of locoregional recurrence of breast cancer after breast conserving surgery. Mammary fat pads of female NOD-SCID gamma mice were each injected with MCF-7 cells in Matrigel. Tumors were allowed to engraft for 2 weeks, after which time either sterile saline (n = 20) or human fat graft (n = 20) was injected adjacent to tumor sites. After 8 weeks, tumors were assessed for volume measurement, histologic grade, Ki67 positivity, and metastatic spread. Animals receiving lipofilling after tumor cell engraftment had lower tumor volume and mass (p = 0.046 and p = 0.038, respectively). Macroscopic invasion was higher in the saline group. Histologic grade was not significantly different in the two groups (p = 0.17). Ki67 proliferation index was lower in tumors surrounded by fat graft (p = 0.01). No metastatic lesion was identified in any animal. Adipose transfer for breast reconstruction performed in the setting of residual breast tumor in a clinically relevant animal model did not increase tumor size, proliferation, histologic grade, or metastatic spread. This study supports the oncologic safety of lipofilling as part of the surgical platform for breast reconstruction after cancer therapy.

Promotion of Primary Murine Breast Cancer Growth and Metastasis by Adipose-Derived Stem Cells Is Reduced in the Presence of Autologous Fat Graft.

Cell-assisted lipotransfer involves enrichment of autologous fat with supraphysiologic numbers of adipose-derived stem cells to improve graft take. Adipose-derived stem cells have been shown to promote cancer progression, raising concerns over the safety of adipose-derived stem cells and cell-assisted lipotransfer in postoncologic breast reconstruction. The authors compared the effect of adipose-derived stem cells alone, cell-assisted lipotransfer, and conventional fat grafting on breast cancer growth and metastasis. Proliferation and migration of murine 4T1 breast cancer cells cultured in control medium or mouse adipose-derived stem cell- or fat graft-conditioned medium were assessed by flow cytometry and scratch assay, respectively. Transcription levels of arginase-1, transforming growth factor-β, and vascular endothelial growth factor were assessed in adipose-derived stem cells and fat graft by quantitative reverse transcription polymerase chain reaction. An orthotopic mouse tumor model was used to evaluate breast cancer progression and metastasis. 4T1 cells were injected into the mammary pad of female BALB/c mice. Six days later, tumors were injected with saline, adipose-derived stem cells, fat graft, or cell-assisted lipotransfer (n = 7 per group). Two weeks later, primary tumors were examined by immunohistochemistry and lung metastasis was quantified. Adipose-derived stem cell-conditioned medium increased cancer cell proliferation (p = 0.03); migration (p < 0.01); and transcription of arginase-1, transforming growth factor-β, and vascular endothelial growth factor compared to fat graft-conditioned or control medium (p < 0.02). Tumor-site injection with adipose-derived stem cells alone led to increased primary tumor growth and lung metastasis compared to control, fat graft, or cell-assisted lipotransfer groups (p < 0.05). Adipose-derived stem cell injection increased CD31 vascular density in tumors (p < 0.01). Adipose-derived stem cells alone, but not conventional fat graft or cell-assisted lipotransfer, promote breast cancer cell proliferation and invasiveness in vitro and in vivo.