Breast Tissue Of Mice Research Articles

Optical parameters of healthy and tumor breast tissues in mice.

Knowledge of the optical parameters of tumors is important for choosing the correct laser treatment parameters. In this paper, optical properties and refraction indices of breast tissue in healthy mice and a 4T1 model mimicking human breast cancer have been measured. A significant decrease in both the scattering and refractive index of tumor tissue has been observed. The change in tissue morphology has induced the change in the slope of the scattering spectrum. Thus, the light penetration depth into tumor has increased by almost 1.5-2 times in the near infrared "optical windows." Raman spectra have shown lower lipid content and higher protein content in tumor. The difference in the optical parameters of the tissues under study makes it possible to reliably differentiate them. The results may be useful for modeling the distribution of laser radiation in healthy tissues and cancers for deriving optimal irradiation conditions in photodynamic therapy.

Maternal obesity driven changes in collagen linearity of breast extracellular matrix induces invasive mammary epithelial cell phenotype

Obesity has been linked with numerous health issues as well as an increased risk of breast cancer. Although effects of direct obesity in patient outcomes is widely studied, effects of exposure to obesity-related systemic influences in utero have been overlooked. In this study, we investigated the effect of multigenerational obesity on epithelial cell migration and invasion using decellularized breast tissues explanted from normal female mouse pups from a diet induced multigenerational obesity mouse model. We first studied the effect of multigenerational diet on the mechanical properties, adipocyte size, and collagen structure of these mouse breast tissues, and then, examined the migration and invasion behavior of normal (KTB-21) and cancerous (MDA-MB-231) human mammary epithelial cells on the decellularized matrices from each diet group. Breast tissues of mice whose dams had been fed with high-fat diet exhibited larger adipocytes and thicker and curvier collagen fibers, but only slightly elevated elastic modulus and inflammatory cytokine levels. MDA-MB-231 cancer cell motility and invasion were significantly greater on the decellularized matrices from mice whose dams were fed with high-fat diet. A similar trend was observed with normal KTB-21 cells. Our results showed that the collagen curvature was the dominating factor on this enhanced motility and stretching the matrices to equalize the collagen fiber linearity of the matrices ameliorated the observed increase in cell migration and invasion in the mice that were exposed to a high-fat diet in utero. Previous studies indicated an increase in serum leptin concentration for those children born to an obese mother. We generated extracellular matrices using primary fibroblasts exposed to various concentrations of leptin. This produced curvier ECM and increased breast cancer cell motility for cells seeded on the decellularized ECM generated with increasing leptin concentration. Our study shows that exposure to obesity in utero is influential in determining the extracellular matrix structure, and that the resultant change in collagen curvature is a critical factor in regulating the migration and invasion of breast cancer cells.

Abstract PS16-18: Inhibition of mTOR signaling by rapamycin abrogates mammary stem/progenitor cell activity in aged mice

Abstract Previous studies have shown an altered physiology in the mammary gland of aged mice, with mammary ducts displaying increased tertiary branching along with an enriched myoepithelial population compared to younger mice. It is the population of myoepithelial cells residing in the basal layer of mammary ducts that is thought to be responsible for the development of the mammary gland. These qualities have been attributed to the mammary stem cells (MaSCs) that reside in this population and are capable of regenerating the mammary gland when implanted in an empty mammary fat pad, indicating their bipotent nature. It is not known however, if these stem cells are responsible for the aging mammary gland phenotype and the increased risk of cancer seen in aging mammals. Our preliminary studies showed that rapamycin could be utilized as a MaSC inhibitor and clinical trials are currently under way investigating the role of rapamycin in inhibiting MaSC function and abrogating biomarkers associated with invasive BC progression. Mammalian target of rapamycin (mTOR) is a protein kinase that regulates growth, proliferation, and survival in cells and is often upregulated in cancer. Rapamycin is an extremely selective inhibitor of mTORC1 function and its downstream signaling. In this study, aged mice fed ad lib with microencapsulated rapamycin showed decreased mTOR activity in the mammary ducts as shown by immunohistochemistry in aged mice. Treated mouse breast tissue also displayed a decreased population of Lin- CD24low CD49fhi myoepithelial/basal cells along with decreased tertiary branching in the ductal morphology, effectively reversing the aged mammary gland phenotypes. MaSC inhibition by rapamycin was also shown using sphere formation efficiency (SFE) assays after sorting the luminal (Lin- CD24hi CD49flow) and basal (Lin- CD24low CD49fhi) epithelial cells using Florescence Activated Cells Sorting (FACS) in a free suspension culture. Together, these findings show a possible use in regulating MaSCs by rapamycin and its potential in preventing age-related diseases such as breast cancer. Citation Format: Larry E. Broome, Hakim Bouamar, James F. Nelson, Lu-Zhe Sun. Inhibition of mTOR signaling by rapamycin abrogates mammary stem/progenitor cell activity in aged mice [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 PS16-18.

S907 DEVELOPMENT OF HUMANIZED MOUSE MODEL FOR STUDYING MOTHER TO CHILD HTLV‐1 TRANSMISSION AND PREVENTION WITH HTLV‐1 ANTIBODY TREATMENT

Background:Human T‐cell leukemia virus type 1 (HTLV‐1) infects mature T cells and causes adult T‐cell leukemia (ATL) and a chronic inflammatory central nervous system disease called HAM/TSP. HTLV‐1 transmissions mainly occurred in children via the breast milk of HTLV‐1+ carrier mothers. Thus, bottle‐feeding or avoiding long term breastfeeding are recommended in the endemic areas of Japan and this resulted in a drastic decrease in new HTLV‐1 infections. Previous studies have shown that approximately 3% of children born from HTLV‐1+ carrier mothers were still infected even when they were exclusively bottle‐fed. This suggested the possible involvement of alternative HTLV‐1 transmission routes, such as placental or birth canal transmission.Aims:Here, we attempted to develop new humanized mouse model of HTLV‐1 mother to child (MTC) transmission for evaluating HTLV‐1 antibody to prevent HTLV‐1 MTC infection.Methods:NOD/SCID/gammac (null) (NOG) pregnant mice were intraperitoneally injected with human PBMCs before being transplanted with a mitomycin C‐treated HTLV‐1‐infected cell line (MT‐2). To evaluate the importance of HTLV‐1 infections via breastfeeding or before birth, we designed a study that switched HTLV‐1‐infected carrier mother (CM) mice with non‐infected healthy mother (HM) mice. HTLV‐1 infection was determined by the real‐time PCR quantification of HTLV‐1 DNA isolated from mouse liver, peripheral blood and breastmilk.Results:Although no HTLV‐1‐infected T cells were detected in the peripheral blood of newborn mice at day 14, HTLV‐1‐infected cells were exclusively observed in the liver tissues via FACS and PCR. Furthermore, highly enriched HTLV‐1+ cells were still detected in mouse breast milk 26 days after birth by PCR. These data suggest that there might be an enrichment system of HTLV‐1 infectious cells in mouse breast tissue. We next performed the mother‐pup pair switching study. After switching, CM mice were maintained with HM mice pups (potentially HTLV‐1−), and HM mice were maintained with CM mice pups (potentially HTLV‐1+). This switching at birth resulted in 28% of the potentially HTLV‐1− pups to become HTLV‐1+, since they were breastfed by an HTLV‐1+ mother. Similarly, 42% of the potentially HTLV‐1+ pups were HTLV‐1+ at 40 days after switching, as they were breastfed by an HTLV‐1− mother. These results suggested that both breastfeeding and canal or placental transmissions presented equal risks with respect to HTLV‐1 infections. We finally analyzed the HTLV‐1 infectious status both in mouse embryo and placenta at E17.5 and found HTLV‐1+ cells both in fetal liver (25%) and placenta (37%).Summary/Conclusion:Together, these data suggest that during pregnancy, HTLV‐1 cells invade the fetal tissues and might be a potential resource of HTLV‐1 infection. We are now trying to assess the effect of HTLV‐1 antibody for preventing MTC HTLV‐1 infection.

Second and third harmonic generation microscopy visualizes key structural components in fresh unprocessed healthy human breast tissue.

Real‐time assessment of excised tissue may help to improve surgical results in breast tumor surgeries. Here, as a step towards this purpose, the potential of second and third harmonic generation (SHG, THG) microscopy is explored. SHG and THG are nonlinear optical microscopic techniques that do not require labeling of tissue to generate 3D images with intrinsic depth‐sectioning at sub‐cellular resolution. Until now, this technique had been applied on fixated breast tissue or to visualize the stroma only, whereas most tumors start in the lobules and ducts. Here, SHG/THG images of freshly excised unprocessed healthy human tissue are shown to reveal key breast components—lobules, ducts, fat tissue, connective tissue and blood vessels, in good agreement with hematoxylin and eosin histology. DNA staining of fresh unprocessed mouse breast tissue was performed to aid in the identification of cell nuclei in label‐free THG images. Furthermore, 2‐ and 3‐photon excited auto‐fluorescence images of mouse and human tissue are collected for comparison. The SHG/THG imaging modalities generate high quality images of freshly excised tissue in less than a minute with an information content comparable to that of the gold standard, histopathology. Therefore, SHG/THG microscopy is a promising tool for real‐time assessment of excised tissue during surgery.

Abstract 3785: Loss of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis

Abstract Vacuolar ATPase (V-ATPase) is expressed intracellularly on vesicular membranes, and controls intracellular pH and acidification of various intracellular vesicles. Most cancer cells express V-ATPase on their cell surface where they appear to acidify the extracellular environment and help cancer cells to metastasize. We have previously demonstrated that the a2 isoform of V-ATPase (a2V) of mammary epithelial cells plays a key role during breast tumor progression and metastasis. However, the role of a2V of hematopoietic stem cells during breast tumor pathogenesis is not known. Therefore, to understand the role of a2V in hematopoietic stem cells during breast tumor progression, we conditionally knocked out a2V (cKO) from the hematopoietic stem cells of mice. We observed that deletion of a2V leads to a significant reduction of CD4+ and CD8+ T cells in the periphery and spleen of the a2V cKO mice compared to control mice. We hypothesized that reduction of helper CD4+ cells, or cytotoxic CD8+ T cells would result in larger and faster-growing tumors in the a2V-cKO mice. After implantation of a syngeneic tumor cell line (E0771) in the breast tissue of mice, we observed that deletion of a2V led to larger and faster-growing breast tumors compared to control mice. Further investigation of the tumor microenvironment revealed that there was a significant reduction in percentage of CD4+ and CD8+ T cells in the a2V-cKO mice compared to control mice. Targeted RNAseq of tumor microenvironment demonstrated that out of 499 genes that were analyzed, only 3 genes were significantly upregulated while 144 genes were significantly downregulated in the a2V cKO mice. The 144 downregulated genes included genes for pro-inflammatory cytokines, T cell specific genes, death receptors and proapoptotic genes. Collectively, these results demonstrate that deletion of a2V from the hematopoietic stem cells leads to a decrease in CD4+ and CD8+ T cells in the periphery, which results in a faster-growing and larger breast tumor. Further study is needed to understand the relative contribution of CD4+ or CD8+ T cells during breast tumor pathogenesis. Citation Format: Manoranjan Sahoo, Gajendra K. Katara, Mahamood Y. Bilal, Sara Fleetwood, Arpita Kulshrestha, Safaa A. Ibrahim, Kenneth D. Beaman. Loss of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3785.

Absence of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis

Abstract Vacuolar ATPase (V-ATPase) is expressed on surface of most cancer cells where it acidifies the extracellular milieu and promotes metastasis. We have previously demonstrated that the a2 isoform of V-ATPase (a2V) of mammary epithelial cells, plays a key role during breast tumor progression and metastasis. However, the role of a2V in hematopoietic stem cells (HSC) during breast tumor progression is not known. Therefore, to understand the role of a2V in HSC during breast tumor progression, we conditionally knocked out a2V (cKO) from the HSC of mice. We observed that deletion of a2V leads to a significant reduction of CD4+ and CD8+ T cells in the spleen of the a2V cKO mice compared to control mice. We hypothesized that the reduction of helper CD4+ cells, or cytotoxic CD8+ T cells would result in larger and faster growing tumors in the a2V-cKO mice. After implantation of a syngeneic tumor cell line (E0771) in the breast tissue of mice, we observed that lack of a2V leads to larger and faster growing breast tumors compared to controls. Further investigation of the tumor microenvironment revealed that there was a significant reduction in percentage of CD4+ and CD8+ T cells in the a2V-cKO mice compared to control mice. Targeted RNAseq of the tumor microenvironment demonstrated that out of 499 genes those were analyzed, only 3 genes were significantly upregulated while 144 genes were significantly downregulated in the a2V cKO mice. The 144 downregulated genes included genes for pro-inflammatory cytokines, T cell specific genes, death receptors and pro-apoptotic genes. Collectively, these results demonstrate that deletion of a2V from the HSC leads to a decrease in CD4+ and CD8+ T cells in the periphery, which results in a faster growing and larger breast tumor.

Abyssinone V-4' Methyl Ether Isolated from Erythrina droogmansiana (Leguminosae) Inhibits Cell Growth and Mammary Glands Hyperplasia Induced in Swiss Mice by the 7,12-Dimethylbenz(a)anthracene.

There is a long standing interest in the identification of medicinal plants and derived natural products for developing cancer therapeutics. The present study was designed to evaluate the in vitro and in vivo antiproliferative effects of Abyssinone V-4' methyl ether (AVME) on breast tissue of mice. The cytotoxicity of AVME was evaluated using MTT assay in four cancer cell lines (DU145, PC3, HepG2, and MCF-7). Further, a protective effect of AVME was evaluated on 7,12-dimethylbenz(a)anthracene- (DMBA-) induced breast tumor in Swiss mice. Incidence, burden, volume, and histological analysis of mammary tumors were measured. As a result, AVME inhibits DU145, PC3, HepG2, and MCF-7 cells growth. In vivo, no tumor was detected in mice from the normal group as compared to those of DMBA group. Moreover, AVME inhibits the DMBA-induced mammary glands hyperplasia in mice at the dose of 10 mg/kg, evidenced by a decrease of tumor incidence, tumor weight, and volume as well as a protective effect against the lobular alveolar hyperplasia. Taken all together, these results suggest that Abyssinone V-4' methyl ether is endowed with antitumor properties and could be a source of traditional medicine which deserves to be more elucidated and explored in the foreseeable future.

Abstract 2507: Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer

Abstract Background: Carboxypeptidase N (CPN) plays an important role in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. Herein we demonstrated that the circulating peptides specifically cleaved by CPN in the tumor microenvironment can indeed be stage-specific indicators of breast cancer. Methods: The activity of CPN was evaluated using an ex-vivo peptide cleavage assay, in which synthesized C3f peptide (His6-C3f_S1304-R1320-His6) is incubated in interstitial fluids of breast tumor and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. Fragment profiling, by an approach combining nanopore fractionation and mass spectrometry, revealed the nature and extent of cleavage by CPN. These results correlated with the level of CPN-catalyzed peptides in blood specimens taken from the tumor-bearing mice, healthy women and breast cancer patients. CPN expression in the same set of samples was further examined by immunohistochemistry and immunoblotting. Results: We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both the mouse and clinical patient samples, the amount of CPN was clearly increased in tumor tissues compared to that seen in normal breast tissue, while its counterpart in blood remained constant. The amount of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n=8) at 2 weeks after orthotropic implantation. Six homologous peptides displayed the significantly higher expression in the patients' plasma as early as the first pathologic stage of breast cancer. Conclusions: The amount of circulating CPN-catalyzed peptides reported here reflects the extent of this enzyme's activity in tumors, and our results clearly indicate their strong potential as biomarkers for non-invasive and early diagnosis of breast cancer. It would certainly be naïve to think that CPN-catalyzed peptides and the enzyme itself are the only biomarkers specific for breast cancer diagnosis. We do however argue that they can be used as a complementary measure for very early diagnosis of tumor growth, particularly because CPN activity within the tumor at this point is already detectable by blood sampling. Such sensitivity together with all of the features mentioned above lend well to their translation to the clinic, for breast cancer and perhaps other pathologies. Citation Format: Ye Hu. Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2507. doi:10.1158/1538-7445.AM2014-2507

P0008 Validating the link between obesity and breast inflammation in women with breast cancer

Background In post-menopausal women, obesity is a risk factor for hormone sensitive breast cancer. We previously described crown-like structures (CLS), consisting of macrophages surrounding dead adipocytes, in the breast tissues of mice and humans. CLS were associated with increased levels of proinflammatory mediators known to be involved in carcinogenesis. We provided the first evidence of CLS in the human breast (CLS-B), which correlated with increased aromatase expression and activity. Here, we expanded our population to prospectively validate these preliminary findings. Methods White adipose tissue (WAT) was prospectively collected from women undergoing breast and reconstructive surgery. WAT was subjected to immunohistochemistry for CD68, a macrophage marker, to detect CLS-B by light microscopy. Adipocyte diameter was measured using the Canvas 11 Software. Endpoints were CLS-B presence/absence and CLS-B index (proportion of slides with CLS-B). Findings From April 2010 to February 2012, WAT (100 mastectomy and five abdominal reconstructions) was obtained from 101 American women; median age 49 years (range 26–80). CLS-B were found in 54 (53%) patients. CLS-B were seen in 9/37 (24%) normal weight patients (body mass index [BMI] μ m) compared with those without CLS-B (91.5 ± 16.1 μm; p p = 0.04). Among 25 patients with bilateral breast WAT and five patients with paired breast and abdominal WAT, concordant CLS-B findings (+/–) were seen in 80%. Interpretation Findings from this prospective study confirm that CLS-B are associated with BMI and adipocyte size. These results provide a plausible pathophysiological link between obesity and breast cancer. Abdominal WAT may be a surrogate for breast WAT inflammation; biopsies of abdominal subcutaneous WAT are more easily done, and could be performed in future clinical trials that aim to decrease breast WAT inflammation.

Circulating Proteolytic Products of Carboxypeptidase N for Early Detection of Breast Cancer

Carboxypeptidase N (CPN) is important in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. We investigated whether circulating peptides specifically cleaved by CPN in the tumor microenvironment can be stage-specific indicators of breast cancer. CPN activity was measured using an ex vivo peptide cleavage assay by incubating synthesized C3f peptide (His6-C3f_S1304-R1320-His6) in interstitial fluids of breast tumors and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. The nature and extent of peptide cleavage by CPN was investigated by fragment profiling using nanopore fractionation and mass spectrometry. The fragment profiles in interstitial fluid correlated with concentrations of CPN-catalyzed peptides in blood samples taken from the tumor-bearing mice, healthy women, and breast cancer patients. CPN expression in the same set of samples was further examined by immunohistochemistry and immunoblotting. We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both the mouse and clinical patient samples, CPN was clearly increased in tumor tissues compared with normal breast tissue, whereas corresponding CPN abundance in blood remained constant. Concentrations of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n = 8) at 2 weeks after orthotopic implantation. Six homologous peptides displayed significantly higher expression in the patients' plasma as early as the first pathologic stage of breast cancer. Circulating CPN-catalyzed peptide concentrations reflect the CPN activity in tumors. These biomarkers show strong potential for the noninvasive and early diagnosis of breast cancer.

Abstract B78: Rebastinib, a small molecule TIE2 kinase inhibitor, prevents primary tumor growth and lung metastasis in the PyMT breast cancer model

Abstract Rebastinib is an inhibitor of tyrosine kinases TIE2 and ABL1. A clinical trial in chronic myeloid leukemia (CML) to inhibit BCR-ABL was previously performed but efficacy was modest and development in CML is not continuing. Rebastinib is approximately 50 times more potent as an inhibitor of TIE2 than BCR-ABL. Biomarker analysis of patients from the CML trial of rebastinib revealed that a majority displayed significant increases in circulating angiopoietin 2 (ANG2) levels. Increases in ANG2, a TIE2 ligand, have been previously reported to correlate with inhibition of the TIE2 pathway. The hypoxic tumor environment engendered by radiation therapy, cytotoxic chemotherapy, or anti-angiogenic treatments (such as anti-VEGF therapies) leads to rebound tumor vascularization by the recruitment of pro-vasculogenic TIE2 expressing monocytes (TEMs) from the bone marrow to these hypoxic tumor sites. TEMs are believed to play an important role in the revascularization of tumors after these treatments, leading to progression of residual tumor. Thus, inhibition of TEM recruitment and activity could lead to better patient outcomes. The polyoma middle-T antigen (PyMT) syngeneic mouse breast cancer model utilizes the mouse mammary tumor virus (MMTV) promoter, a breast specific promoter, to express PyMT in mouse breast tissue. In this model, primary breast cancers spontaneously occur, proliferate, and metastasize (mainly to the lungs) and lead to the death of the mice. Unlike xenografts, the PyMT model utilizes fully immunocompetent mice and metastasis in this model is known to be modulated by TEMs. Rebastinib has been evaluated in a PyMT mouse where primary tumors were allowed to reach 800 mg in size before starting treatment. Rebastinib therapy resulted in a significant decrease in the growth rate of the primary breast tumor (75%), a 71% reduction in lung metastases, a decrease in the levels of tumoral TIE2 staining by immunohistochemical analysis (IHC) and caused the remaining tumor to become necrotic. Combining rebastinib with paclitaxel resulted in a synergistic response with 90% inhibition of tumor growth and a 93% reduction of lung metastases. This presentation will focus on the biochemical, cellular and in vivo activity of rebastinib as a novel small molecule inhibitor of TIE2 kinase and its potential for prevention of primary tumor rebound and metastasis in combination with first line interventional therapy. A clinical trial exploring this activity is planned for 2013. Citation Format: Bryan D. Smith, Molly M. Hood, Michael D. Kaufman, Mark Berger, Daniel L. Flynn, Scott C. Wise. Rebastinib, a small molecule TIE2 kinase inhibitor, prevents primary tumor growth and lung metastasis in the PyMT breast cancer model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B78.

Abstract B102: Fibroblast-dependent modulation of drug sensitivity, stemcellness, and tumor cell dormancy

Abstract Solid tumors are composed of tumor cells and different cell types making up the tumor stroma — endothelial cell, pericytes, immune cells and cancer associated fibroblasts. We focus on the impact of cancer associated fibroblasts on the stem cell properties of tumor cells, including drug sensitivity, anchorage independent growth and tumor initiating capacities. Previous work in our group showed that expression of platelet derived growth factor receptor β (PFGFRβ) in the tumor stroma is associated with worse prognosis in breast cancer. Furthermore, two experimental approaches were used to explore PDGFR-dependent paracrine signaling: (1) primary human breast fibroblasts isolated from normal as well as cancer tissue co-cultured with human breast cancer cell lines and (2) tumor cells, cancer associated fibroblasts and normal breast tissue fibroblasts derived from a genetic breast cancer mouse model. When looking at PFGFRβ expression in tumors from a randomized tamoxifen study we found that tumors with high expression in the tumor stroma did not seem to respond to the treatment. Ongoing in vitro studies are analyzing the impact of co-cultured fibroblasts on breast cancer cell tamoxifen sensitivity in vitro. Specifically we want to investigate how PDGF receptors, expressed by the tumor stroma, play a role in the sensitivity to tamoxifen by the tumor cells. We have also investigated the impact of fibroblasts on stemness properties of tumor cells derived from tumors of the MMTV polyoma middle T antigen driven mouse breast cancer model. Conditioned medium of tumor associated fibroblasts but also from normal mouse breast tissue fibroblasts increased the anchorage independent sphere forming capacity of the tumor cells in vitro. Conditioned media from the tumor cells themselves had similar effects. Tumor-supporting effects of fibroblasts were confirmed after orthotopic coinjection of tumor cells together with fibroblasts in syngeneic mice. The presence of fibroblasts resulted in a significant increase in the tumor take rate. Together these studies identify fibroblast-mediated modulation of “stemness” of breast cancer cells and a PDGFR-dependent modulation by fibroblasts of tamoxifen-sensitivity. Citation Format: Janna Paulsson, Carina Strell, Pernilla Roswall, Helena Rannikmae, Anne Weiland, Lisa Rydén, Arne Östman. Fibroblast-dependent modulation of drug sensitivity, stemcellness, and tumor cell dormancy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B102.

Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.

Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.

Abstract P5-10-17: Radiotherapy-induced miR expression influences the formation of local recurrence in breast cancer

Abstract In early breast cancer (EBC) patients, local disease relapse occurs at the site of the original primary tumor in 90% of cases and the addition of external beam radiotherapy (EBRT) after surgery is necessary to reduce recurrence rate. This observation suggested that surgery itself could represent a perturbing factor, possibly by altering the local microenvironment by inducing a wound healing response. Recent evidences indicate that, in selected EBC patients, 5 weeks of EBRT can be successfully replaced by a single dose of Targeted Intraoperative Radiotherapy (TARGIT). Interestingly, TARGIT acts not only affecting tumor cell survival itself, but is also able to alter the tumor microenvironment, by modifying cytokines secretion and activation of several intracellular pathways in breast cancer cells. In order to understand the significance of these modifications in breast tumor microenvironment after exposure to TARGIT we set up a mouse model of TARGIT treatment. Our results highlighted that the act of surgery alone strongly stimulates breast cancer cell growth and the addition of local irradiation completely reverses the surgery-induced breast cancer cell growth. Moreover, we evaluated the impact of TARGIT by analyzing microRNA (miR) expression both in our mouse model and in human specimens. To this aim, we collected paired specimens of peri-tumoral mammary tissues from 30 early breast cancer (EBC) patients, before and after treatment with TARGIT. Microarray approach followed by qRT-PCR validation revealed the presence of specific TARGIT-regulated miRs. Among the others, miR-223 was the most highly and significantly modified. In silico analyses indicated that epidermal growth factor (EGF) is a potential candidate of mR-223 regulation. In vitro experiments, using both normal and tumor derived cell lines validated the bioinformatic prediction, demonstrating that the 3′ UTR of EGF is a target of miR-223 and this binding results in the control of EGF production by the cells. Importantly, miR-223 overexpression was able to impair, at least in part, the wound-induced growth of normal and tumor derived breast-cancer cells, eventually influencing breast cancer cell proliferation and survival. Our results suggest that the effects of radiotherapy in the wounded tissue go far beyond the mere killing of residual tumor cells and is able to balance the negative effects of the wound healing process, both in human and mouse breast tissue. TARGIT-induced microenvironment modifications, in particular miR-223 up-regulation, significantly impair breast cancer response to surgery-induced inflammation eventually resulting in recurrence control. Our experimental findings strongly support the notion that the timely application of radiotherapy to the tumor bed, immediately after tumor removal, is critical to counteract the effects of surgery and wound healing process on residual breast cancer cells and normal peri-tumoral breast tissue. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-17.

Abstract LB-93: The role of GSK-3 in mammary gland development and oncogenesis

Abstract Glycogen synthase kinase 3, comprised of two isoforms: α and β, is a central regulator of several signaling pathways that play key roles in development and cancer. Chemical inhibition or genetic loss of GSK-3 in a normal mouse breast cell line cultured under conditions resembling the physiological breast environment resulted in morphological defects reminiscent of early stages of disease. A similar phenotype was also observed upon expression of an activated allele of β-catenin, a major GSK-3 target protein often mutated and elevated in human cancers. These findings implicate GSK-3 as a key regulator of normal ductal development, which may be disrupted early in cancer progression. Upon genetic deletion of GSK-3 (double knock-out of all four alleles, DKO) from breast tissues of mice, dams are unable to nurse their pups. These animals develop severe mammary intraepithelial neoplasia and squamous metaplasia frequently at first pregnancy. Following 4–6 months of continuous breeding, breast adenocarcinomas are observed with elevated levels of ß-catenin and Gli, along with increased expression of a putative mammary progenitor cell marker. Increased cell growth as well as expression of smooth muscle actin, a myoepithelial marker, are also features of these tumors. Similar developmental and functional defects are also observed in mice with breast glands lacking GSK-3 as well as ß-catenin (triple knock-out, TKO). The epithelium of TKO mammary glands undergoes various degrees of squamous differentiation and keratinization, although at a lower penetrance than the DKOs. Multiparious TKOs develop tumors, albeit with a longer latency (7–9 months). Preliminary flow cytometry analysis of mammary glands from DKO and TKO animals show the ratio of luminal to basal cell populations is increased in absence of GSK-3. We are currently investigating how GSK-3 may regulate mammary stem/progenitor cell commitment to these mammary cell lineages. Previously reported expression of stabilized ß-catenin in the mammary epithelium induces squamous metaplasia, but no adenocarcinomas were observed at 6 months. In our hands, these animals do develop small tumors at ∼10 months of age. These data suggest the observed DKO and TKO phenotypes are not solely mediated via aberrant ß-catenin signaling and that GSK-3 has ß-catenin-independent functions in mammary gland disease. Other pathways regulated by GSK-3 including Notch, Hedgehog and PI3K are being interrogated in DKO and TKO tumor-initiating cells to gain a molecular understanding of tumor development. 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 LB-93. doi:10.1158/1538-7445.AM2011-LB-93

Abstract 2368: Autotaxin/Enpp2 controls of bone metastasis formation through osteoclast function

Abstract Autotaxin (ATX, NPP-2) is a secreted protein with both an oncogenic action on mouse breast tissue and metastatic properties on breast cancer cells. Due to its lysophospholipase D activity, ATX generates lysophosphatidic acid (LPA) from lysophosphatidylcholine and controls LPA levels in the blood. We have recently showed that platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. However, how LPA is produced at the metastatic site and the role of ATX by cancer cells in this metastasis process are still unknown. Here, we found that in vitro, forced expression of ATX in human breast cancer MDA-B02 cells (MDA-B02/ATX) increased invasiness, proliferation and enhanced their pro-osteoclastic function. In vivo, intravenous injection of MDA-B02/ATX cells to inmmunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss and tumor burden, and higher active osteoclast number at the bone metastatic site. Mouse 4T1 breast cancer cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. We found that 4T1 cells expressed active ATX. In vitro, silencing ATX[O1] expression inhibited invasion but not proliferation of 4T1-siATX cells. In vivo, silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at bone the metastatic site. Altogether, these results demonstrated that expression of ATX by cancer cells controls bone metastasis formation and that targeting ATX in patients might contribute to the development of additional therapies against bone metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2368.

Expression and Secretion of Cathelicidin Antimicrobial Peptides in Murine Mammary Glands and Human Milk

Mammalian milk possesses inherent antimicrobial properties that have been attributed to several diverse molecules. Recently, antimicrobial peptides that belong to the cathelicidin gene family have been found to be important to the mammalian immune response. This antimicrobial is expressed in several tissues and increased in neonatal skin, possibly to compensate for an immature adaptive immune response. We hypothesized that the mammary gland could produce and secrete cathelicidin onto the epithelial surface and into milk. Human cathelicidin hCAP18/LL-37 mRNA was detected in human milk cells by PCR. Quantitative real-time PCR demonstrated an increase in relative expression levels at 30 and 60 d after parturition. Immunohistochemistry of mouse breast tissue identified the murine cathelicidin-related antimicrobial peptide in lobuloacinar and ductules. Western blot analysis of human milk showed that LL-37 was secreted and present in the mature peptide form. The antimicrobial activity of LL-37 against Staphylococcus aureus, group A Streptococcus, and enteroinvasive Escherichia coli O29 in the human milk ionic environment was confirmed by solution colony-forming assay using synthetic peptide. These results indicate that cathelicidin is secreted in mammary gland and human milk, has antimicrobial activity against both Gram-positive and Gram-negative bacteria, and can contribute to the anti-infectious properties of milk.

Reconstruction of functionally normal and malignant human breast tissues in mice.

The study of normal breast epithelial morphogenesis and carcinogenesis in vivo has largely used rodent models. Efforts at studying mammary morphogenesis and cancer with xenotransplanted human epithelial cells have failed to recapitulate the full extent of development seen in the human breast. We have developed an orthotopic xenograft model in which both the stromal and epithelial components of the reconstructed mammary gland are of human origin. Genetic modification of human stromal cells before the implantation of ostensibly normal human mammary epithelial cells resulted in the outgrowth of benign and malignant lesions. This experimental model allows for studies of human epithelial morphogenesis and differentiation in vivo and underscores the critical role of heterotypic interactions in human breast development and carcinogenesis.