Mammary Gland Fat Pad Research Articles

Establishment of Translational Luciferase-Based Cancer Models to Evaluate Antitumoral Therapies.

Luciferase (luc) bioluminescence (BL) is the most used light-emitting protein that has been engineered to be expressed in multiple cancer cell lines, allowing for the detection of tumor nodules in vivo as it can penetrate most tissues. The goal of this study was to develop an oncolytic adenovirus (OAd)-resistant human triple-negative breast cancer (TNBC) that could express luciferase. Thus, when combining an OAd with chemotherapies or targeted therapies, we would be able to monitor the ability of these compounds to enhance OAd antitumor efficacy using BL in real time. The TNBC cell line HCC1937 was stably transfected with the plasmid pGL4.50[luc2/CMV/Hygro] (HCC1937/luc2). Once established, HCC1937/luc2 was orthotopically implanted in the 4th mammary gland fat pad of NSG (non-obese diabetic severe combined immunodeficiency disease gamma) female mice. Bioluminescence imaging (BLI) revealed that the HCC1937/luc2 cell line developed orthotopic breast tumor and lung metastasis over time. However, the integration of luc plasmid modified the HCC1937 phenotype, making HCC1937/luc2 more sensitive to OAdmCherry compared to the parental cell line and blunting the interferon (IFN) antiviral response. Testing two additional luc cell lines revealed that this was not a universal response; however, proper controls would need to be evaluated, as the integration of luciferase could affect the cells' response to different treatments.

Carryover effects of maternal late-gestation heat stress on granddaughters' growth and mammary gland development

Maternal (F0) exposure to late-gestation heat stress reduces their daughter's (F1) mammary gland fat pad mass (FP), parenchyma (PAR) mass, and epithelial cell proliferation when evaluated at birth and weaning, and go on to produce less milk in their first lactation. Herein, we investigated the effect of maternal late-gestation heat stress on whole-body growth and mammary development of their granddaughters (F2). Multiparous F0 cows had access to heat abatement (n = 41, shade, and active cooling via fans and water soakers) or not (n = 41, shade only) for the last 56 d of gestation during a subtropical summer. Consequently, the F1 daughters, born to F0 cows, were heat-stressed (HTF1, n = 36) or cooled (CLF1, n = 37) in utero during the last 2 mo of gestation. All F1 heifers were raised as an identically managed cohort until first calving. The F2 granddaughters, born to HTF1 (HTF2, n = 12) or CLF1 (CLF2, n = 17), were raised as an identically managed cohort until 70 d of age. Dry matter intake (DMI), body weight, hip height, wither height, chest girth, head circumference, mammary gland teat length, and left-right and front-rear teat distances were measured. Average daily gain (ADG) was calculated for the pre-weaned period (0-49 d). Mammary ultrasounds were performed on d 21, 49, and 70 (n = 9/group) on the rear left and right quarters to quantify PAR and FP areas. Mammary biopsies were collected for histological evaluation of epithelial structures (H&E staining), and to quantify cells positive for ERα (estrogen receptor, α subunit), cell proliferation (Ki67), and apoptosis (TUNEL). Heifer growth from birth to d 49 was similar between CLF2 and HTF2 for all parameters evaluated. Distances between teats and teat length were not different between groups. On d 70, CLF2 tended to have a greater average PAR (right and left quarters) relative to HTF2. Although the left FP was smaller in HTF2 relative to CLF2, the average FP was not different. The lumenal and non-lumenal epithelial structures in the PAR of HTF2 were significantly smaller than those of CLF2. In addition, HTF2 had a reduced percentage of proliferating cells in the epithelial and stromal compartments and a greater percentage of apoptotic cells, particularly in the stroma. The percentage of ERα positive cells was significantly reduced in HTF2. In summary, although HTF2 heifer's DMI was similar and they grew at the same rate as CLF2 heifers throughout the pre-weaning phase, their mammary glands had smaller PAR areas with fewer epithelial structures characterized by reduced cell turnover and lower ERα expression. These early changes in the microstructure and cellular turnover of the mammary gland may partly explain the reduction in lactation performance relative to CLF2 counterparts at maturity.

Abstract 167: Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy

Abstract Introduction: Statins, known as inhibitors of HMG-CoA, not only reduce cholesterol but also exhibit pleiotropic effects which include anti-tumor activities by targeting tumor microenvironments. They inhibit the synthesis of FPP and GGPP, which is important for prenylation of signaling proteins. Although PD-1/PD-L1 inhibitors has been approved for treatment for TNBC patients with PD-L1 positive, the response remains unsatisfactory. Also, it has been reported that statins affect PD-L1 expression in other tumors, but it is still uncertain in TNBC. Therefore, we aimed to identify the effect of statins on the expression of PD-L1 in TNBC and to verify the efficacy of combination therapy with statins and immune checkpoint inhibitors. Methods: Human and mouse TNBC cell lines, mouse macrophage cell line (RAW 264.7), and clinically approved simvastatin were used. Flow cytometry, western blot, proliferation assay, qRT-PCR, immune phenotyping and immunohistochemistry were conducted. A co-culture of macrophages and cancer cells was performed. An orthotopic syngeneic mouse model by injection of EMT6 cells into mammary gland fat pad were produced. Mice were treated with simvastatin(20mg/kg) by oral gavage daily and anti-PD-1(200μg) by intraperitoneal injection two times per week. Results: Among thirteen human TNBC cell lines, we confirmed that TNBC cell lines showed various PD-L1 expression, and especially MDA-MB-231 and HCC38 highly expressed endogenous/constitutive PD-L1. Statins reduced PD-L1 expression and exerted anti-proliferative effects in MDA-MB-231, HCC38, and EMT6 in a dose- and time-dependent manner. Phosphorylation of AKT and STAT3 was reduced when simvastatin was treated. Mice with combination therapy of anti-PD-1 and simvastatin had less effective inhibitory effect on tumor growth than mice with anti-PD-1 monotherapy, which showed the highest tumor suppression effect. In immune phenotyping, tumor-infiltrated T cells did not have significant differences among groups. However, M1:M2 macrophage ratio was highest in anti-PD-1 group but decreased in combination therapy group with simvastatin. To determine the effect of simvastatin on macrophages, RAW264.7 cells were stimulated with MDA-MB-231 conditioned medium, revealing an upregulation of PD-1 expression. Conclusions: Our findings show that simvastatin has an anti-tumor effect, which reduces Pd-L1 expression and kills breast cancer cells, reducing phosphorylation of STAT3 and AKT. However, when combined with PD-1 checkpoint inhibitor, it reduces the anti-tumor effect by increasing PD-1 expression in macrophages and promoting M1 to M2 polarization. Further study is needed to demonstrate the mechanism by which simvastatin regulate polarization of macrophages and to confirm the polarization of macrophages in samples of TNBC patients who had been taking simvastatin. Citation Format: Sangeun Lee, Ju Hee Kim, A Young Park, Jihong Baik, Ji-Jung Jung, Han-Byoel Lee, Wonshik Han. Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 167.

Abstract 2635: N-Myristolytransferase (NMT) inhibitors as novel potent payloads for antibody drug conjugates

Abstract Introduction: There is a need for ADC payloads with an improved therapeutic index, consequently we have coupled potent and selective inhibitors of NMT to trastuzumab to test their potential in both in vitro and in vivo models. Experimental Procedures: A highly potent NMT inhibitor was conjugated to trastuzumab using two different linkers to produce an ADCs with a DAR of 5. These ADCs were tested in vitro in cell killing studies using Her2+ and Her2- cell lines and subsequently tested in vivo in breast cancer and gastric cancer xenografts. Results: Our first Trastuzumab-NMT inhibitor ADC (MYX2449) caused cell death in Her2+ BT474 cells with an EC50 of 0.2nM and peaking after 8 days of incubation, but was inactive against Her2- cells MCF7 at concentrations up to 100nM. MYX2449 was tested in a BT474 xenograft implanted orthotopically in the mammary gland fat pad and compared to Trastuzumab and vehicle controls. Both antibodies were dosed IV once a week for four weeks at 2.5 and 5 mg/Kg. MYX2449 therapy at 2.5mg/Kg resulted in a partial response (TGI 55% on day 21) and 5mg/Kg resulted in a TGI of 108% on day 21 with 7/10 mice having undetectable tumor by day 33 with no significant body weight loss during the study. Trastuzumab resulted in no response at 2.5mg/Kg a partial response at 5mg/Kg. MYX2449 was subsequently tested in a gastric cancer xenograft, NCI-N87 inoculated subcutaneously and compared to an NMT inhibitor conjugated isotype control and to Trastuzumab and Trastuzumab deruxtecan. All antibodies were dosed IV at 2.5mg/Kg or 5mg/Kg on day 1 and day 8. Day 21 TGI for the 5mg/Kg Trastuzumab group was 193%, for the 5mg/Kg Trastuzumab-deruxtecan group TGI was 154% and for the 5mg/Kg MYX2449 group TGI was 280%. In the MYX2449 5mg/Kg group, 6/10 mice had undetectable tumors on day 21 and 2/10 of the MYX2449 2.5mg/Kg group. Mean body weights of all groups had increased by day 21. Conclusions: Potent small molecule inhibitors of NMT can be readily conjugated to therapeutic monoclonal antibodies to generate novel highly effective and well tolerated ADC payloads. NMT inhibitors represent a totally novel class of ADC payloads that exploit cancer cell dependency on myristloylated proteins. The potential of this novel mechanism to deliver high efficacy with better TI is currently being investigated in preclinical models. Examples of Myricx patented chemical structures will be disclosed. RS, ND, FF and RC are employees of Myricx Pharma.JW, FO and ET are funded by Myricx Pharma.ET is a board member and founder of Myricx Pharma. Citation Format: Robin A. Carr, Roberto Solari, Nikki D'Arcy, Ed Tate, Josephine Walton, Folake Orafidiya. N-Myristolytransferase (NMT) inhibitors as novel potent payloads for antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2635.

Abstract P5-13-06: Overexpression of somatic mutation in NSDHL promotes breast cancer cell migration and tumor growth

Abstract Background: We had identified somatic mutations of the NAD(P) Dependent Steroid Dehydrogenase-Like (NSDHL) gene from breast tumors of patients with distant metastasis using whole-exome sequencing. This study aimed to investigate the function of NSDHL mutants in breast cancer. Methods: ER-positive and tamoxifen-resistant human breast cancer cell line (ZR75-1) and mouse macrophage cell line (RAW264,7) were used. GFP-tagged NSDHL mutants were generated by subcloning the mutant NDHL into a lentiviral vector yielding an in-frame fusion of 3′ to GFP. ZR75-1 cells expressing the GFP-tagged NSDHL mutants were selected by puromycin with subsequent FACS analysis. The following experiments were performed: western blot, immunofluorescence staining, qRT-PCR, CellTiter-Glo assay, cell cycle assay, transwell migration assay, wound healing assay, tumor spheroid formation assay, and total cellular cholesterol measurement. An orthotopic breast tumor mouse model by injection of ZR75.1 cells into mammary gland fat pad was used in vivo. Results: Four novel NSDHL somatic mutations were discovered in the breast tumor tissues of patients. While total cholesterol levels in NSDHL mutant-transduced cells did not significantly increase, they were notably higher in wild-type NSDHL-transduced cells than in parent ZR75-1 cells. When compared to the parent and wild-type NSDHL-transduced cells, there was a minor increase in growth rate and a large increase in migratory capacities in NSDHL mutant-transduced cells (P< 0.05). The size of tumor spheroids is significantly larger in NSDHL mutant-transduced cells than in wild-type NSDHL-transduced cells. Both EGFR expression and epithelial to mesenchymal transition (EMT) markers were higher in NSDHL mutant-transduced cells than in parent and wild-type NSDHL-transduced cells. The NSDHL mutant-transduced cells’ conditioned media promoted the migratory ability and M2 polarization of RAW264.7 cells. In an orthotopic xenograft mouse model, NSDHL mutant-transduced cells were shown to promote tumor growth better than parent and wild-type NSDHL-transduced cells, suggesting that somatic mutation of the NSDHL contributes to the malignant progression of breast cancer cells. Conclusion: The present data indicate that breast cancer cells harboring somatic mutants of the NSDHL gene seem to display more aggressive behavior by gaining biological worse phenotype both in vitro and in vivo. More investigation is required into the mechanisms behind the function and accumulation of mutant NSDHL proteins to speed the development of therapies for patients with breast cancer harboring mutant NSDHL. Citation Format: Moonjou Baek, Hoe Suk Kim, So-Hyun Yoon, Seungyeon Ryu, Sangeun Lee, Kyoungsook Park, Han-Byoel Lee, Wonshik Han. Overexpression of somatic mutation in NSDHL promotes breast cancer cell migration and tumor growth [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-06.

Abstract P2-26-10: Statins exhibit an anti-tumor effect by attenuating PD-L1 in breast cancer cells and macrophages and reducing breast tumor progression in xenograft mouse model

Abstract Background: Statins were suggested for repurposed drugs, having multifaceted effects which include anti-tumor activities by modulating the immune response. Here, we aimed to demonstrate the effect of statins on programmed death-ligand 1 (PD-L1) expression in triple-negative breast cancer (TNBC) cells. Methods: Thirteen human TNBC cell lines, mouse macrophage cell line (RAW 264.7), cholesterol and 27-hydroxycholesterol, and clinically approved lovastatin and simvastatin were used. Flow cytometry, Annexin V/propidium iodide assay, western blot, qRT-PCR, transwell migration assay, and immunohistochemistry were employed. A co-culture of macrophages with various breast cancer cells was performed. An orthotopic breast tumor mouse model and metastasis model by injection of GFP-tagged MDA-MB-231 cells into mammary gland fat pad and the tail vein injection were produced. In tumor model mice, lovastatin(10mg/kg) was daily injected intraperitoneally. In vivo fluorescent imaging was used to identify primary tumor development and lung metastasis. Results: Among thirteen TNBC cell lines, MDA-MB-231, HCC38, and HCC70 highly expressed endogenous/constitutive PD-L1. Statins reduced PD-L1 expression and exerted anti-proliferative and apoptotic effects in MDA-MB-231, HCC38, HCC70, and Raw264.7 in a dose- and time-dependent manner (p< 0.05). Meanwhile, statins increased the expression of PD-L1 in Hs578T and MDA-MB-468. STAT3 phosphorylation was inhibited in MDA-MB-231 and HCC70, but not in Hs578T, while AKT phosphorylation was reduced in MDA-MB-231, HCC70, Hs578T, and MDA-MB-468 when statins were treated. The migration of MDA-MB-231 and Raw264.7 in statin-treated conditioned media was decreased (p< 0.05). Cholesterol and 27-hydroxy cholesterol did not restore PD-L1 expression in statin-treated MDA-MB-231. Statins suppressed the expression of M2 markers (PD-L1, CD206, YM-1, Fizz1, arginase-1) in RAW26437 stimulated by a conditioned medium of MDA-MB-231. Lovastatin suppressed the primary tumor growth and metastasis in xenograft tumor mice. Conclusions: Our findings show that statins have an anti-tumor effect, which kills breast cancer cells and triggers macrophage reprogramming by reducing PD-L1 expression, impairing the AKT, ERK, and STAT3 signal pathways, and decreasing M2 markers. Further study is needed to investigate an in-depth molecular mechanism study by which statins regulate PD-L1 expression in TNBC and to confirm the safe and effective use of statins as adjuvant therapy in TNBC. Citation Format: Sangeun Lee, Hoe Suk Kim, So-Hyun Yoon, Seungyeon Ryu, Moonjou Baek, A Young Park, Han-Byoel Lee, Wonshik Han. Statins exhibit an anti-tumor effect by attenuating PD-L1 in breast cancer cells and macrophages and reducing breast tumor progression in xenograft mouse model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-10.

Tissue-resident macrophages are major tumor-associated macrophage resources, contributing to early TNBC development, recurrence, and metastases

Triple-negative breast cancer (TNBC) is an aggressive and highly heterogenous disease with no well-defined therapeutic targets. Treatment options are thus limited and mortality is significantly higher compared with other breast cancer subtypes. Mammary gland tissue-resident macrophages (MGTRMs) are found to be the most abundant stromal cells in early TNBC before angiogenesis. We therefore aimed to explore novel therapeutic approaches for TNBC by focusing on MGTRMs. Local depletion of MGTRMs in mammary gland fat pads the day before TNBC cell transplantation significantly reduced tumor growth and tumor-associated macrophage (TAM) infiltration in mice. Furthermore, local depletion of MGTRMs at the site of TNBC resection markedly reduced recurrence and distant metastases, and improved chemotherapy outcomes. This study demonstrates that MGTRMs are a major TAM resource and play pivotal roles in the growth and malignant progression of TNBC. The results highlight a possible novel anti-cancer approach targeting tissue-resident macrophages.

Gestational and Lactational Exposure to the Emergent Alternative Plasticizer 1,2-Cyclohexane Dicarboxylic Acid Diisononyl Ester (DINCH) Impairs Lipid Metabolism to a Greater Extent Than the Commonly Used Di(2-Ethylhexyl) Phthalate (DEHP) in the Adult Rat Mammary Gland.

Due to their endocrine disruption properties, phthalate plasticizers such as di(2-ethylhexyl) phthalate (DEHP) can affect the hormone-dependent development of the mammary gland. Over the past few years, DEHP has been partially replaced by 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) which also have potential endocrine disrupting properties. The goal of the present study is to understand the impact of a gestational and lactational exposure to DEHP and DINCH on mammary gland development using Sprague Dawley rats. Both plasticizers altered the adipocytes of the mammary gland fat pad of adult progeny, as demonstrated by a decrease in their size, folding of their membrane, and modulations of the lipid profiles. DEHP treatments decreased the expression of Rxrα and Scd1 at the low and high dose, respectively, but did not affect any of the other genes studied. DINCH modulation of lipid metabolism could be observed at puberty by a decreased expression of genes implicated in triglyceride synthesis, lipid transport, and lipolysis, but by an increased expression of genes of the β-oxidation pathway and of genes involved in lipid storage and fatty acid synthesis at adulthood, compared with control and DEHP-treated rats. A strong upregulation of different inflammatory markers was observed following DINCH exposure only. Together, our results indicate that a gestational and lactational exposure to DINCH has earlier and more significant effects on lipid homeostasis, adipogenesis, and the inflammatory state of the adult mammary gland than DEHP exposure. The long-term consequence of these effects on mammary gland health remained to be determined.

Abstract 1350: The role of phosphatidylserine scramblases in tumor progression

Abstract Introduction: Phosphatidylserine (PS), an anionic phospholipid, is asymmetrically distributed in the inner leaflet of the phospholipid bilayer in normal cells under homeostasis. However, PS is constitutively externalized in the tumor microenvironment (TME); on viable cells that undergo stress, such as hypoxic tumor cells, stressed tumor vascular endothelial cells, as well as on cells undergoing apoptosis. This high PS harbors a pro-tumorigenic micro-environment, as myeloid cells expressing PS receptors (Mertk, Tyro3 and Axl) engage with externalized PS and secrete immunosuppressive cytokines. PS is externalized on viable cells by a calcium activated scramblase TMEM16F and on apoptotic cells by a caspase activated scramblase called Xkr8. Here, to study the mechanisms by which PS is externalized in the TME, we hypothesize that TMEM16F and Xkr8 are the major scramblases responsible for creating an immune suppressive milieu in the tumor microenvironment. Methods: To study the mechanisms of PS dysregulation in the tumor microenvironment, TMEM16F and Xkr8 were knocked out in E0771 murine breast cancer cell line using CRISPR/Cas9. The knockouts were validated by western blotting, q-RT-PCR and surveyor assays. Results: TMEM16F KO E0771 cells express lower basal levels of PS compared to WT E0771 cells when detected by Annexin V staining by flow cytometry. TMEM16F KO cells are also less responsive to activation by calcium ionophore. TMEM16F KO, Xkr8 KO and WT E0771 cells have similar viability and migration patterns in response to Gas6, indicating that the cells themselves do not have altered oncogenic potential. TMEM16F KO E0771 cells injected into the inguinal mammary gland fat pad of C57BL6 syngeneic mice showed modest but significantly reduced tumor growth compared to the E0771 WT tumors, suggesting that TMEM16F mediated PS externalization could play a role in exacerbating tumor progression. Conclusion: TMEM16F driven PS externalization contributes to tumor growth. The effect of TMEM16F KO on immune regulation of the tumor milieu will be discussed. These data corroborate previous studies targeting PS in tumors therapeutically using anti-PS antibodies and suggests a role for scramblases in pleiotropic immune-suppression in the tumor. Citation Format: Varsha Gadiyar, Viralkumar Davra, David Calianese, Kevin Lahey, Raymond B. Birge. The role of phosphatidylserine scramblases in tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1350.

Antitumor Immunity from Abdominal Flap-Embedded Therapeutic Cancer Vaccine.

BackgroundAbdominal flaps are routinely performed in clinic after primary mastectomy of breast cancer. However, cancer patients can still develop cancer recurrence and metastasis after surgery. In this study, we evaluated the feasibility of concurrent abdominal flap reconstruction and vaccine inoculation in the tissue for prevention and treatment of HER2-positive breast cancer.MethodsA murine model of metastatic HER2-positive breast cancer was generated by inoculating HER2-expressing TUBO tumor cells into both the mammary gland fat pad and left ventricle. Mammary gland fat pad with primary tumor was resected by mastectomy, and superficial inferior epigastric (SIE) vessel-based abdominal flap was performed for abdominal reconstruction. During the surgery, mice also received a single intra-flap treatment of a microparticulate-based cancer vaccine. Popliteal (Pop) and inguinal (Ing) lymph nodes (LN) were collected at different time points after vaccination, and activation of dendritic cells and T lymphocytes was evaluated with flow cytometry. ELISpot was also performed to measure HER2-specific T cells in splenocytes. In addition, infiltration of CD3+ T cells in brain metastatic nodules was analyzed with immunohistochemistry.ResultsFlow cytometry detected increased number of activated dendritic cells in lymph nodes in mice treated with cancer vaccine. ELISpot revealed abundant IFN-γ-expressing T cells in the spleen. Mice treated with abdominal flap-embedded cancer vaccine extended median survival by 9 days over the control group (p<0.05).ConclusionAbdominal flap-embedded cancer vaccine effectively stimulated systemic immune response and inhibited tumor progression in a murine model of HER2-positive breast cancer.

Application of immunotherapy based on dendritic cells stimulated by tumor cell-derived exosomes in a syngeneic breast tumor mouse model

We here evaluated the therapeutic effect of tumor cell-derived exosomes (TEXs)-stimulated dendritic cells (DCs) in a syngeneic orthotopic breast tumor model. The DC line DC2.4 and breast cancer cell line E0771 originally isolated from C57BL/6 mice were used. E0771 cells stably expressing the exosomal CD63-RFP or luciferase (Luc) and DC2.4 cells stably expressing GFP were produced using lentivirus. TEXs were purified from conditioned medium of E0771/CD63-RFP cells. Breast tumor model was established by injecting E0771/Luc cells into mammary gland fat pad of mice. TEXs contained immune modulatory molecules such as HSP70, HSP90, MHC I, MHC II, TGF-β, and PD-L1. TEXs were easily taken by DC2.4 cells, resulting in a significant increase in the in vitro proliferation and migration abilities of DC2.4 cells, accompanied by the upregulation of CD40. TEX-DC-treated group exhibited a decreased tumor growth compared with control group. CD8+ cells were more abundant in the tumors and lymph nodes of TEX-DC-treated group than in those of control group, whereas many CD4+ or FOXP3+ cells were localized in those of control group. Our results suggest a potential application of TEX-DC-based cancer immunotherapy.

Evaluation of Radiotherapy-Induced Systemic Antitumor Effects in Mice Bearing 4T1 Mouse Breast Cancer Cells.

Background: Recently, several clinical studies have reported that combination treatments of radiation therapy (RT) and immunotherapy in patients with multiple lesions can improve tumor regression at a distance from the irradiated site, known as the abscopal effect. However, when RT and immunotherapy are concurrently applied, it is hard to distinguish the pure systemic effects of RT from those of the immunotherapy drug. In this preclinical study, the authors investigated the systemic antitumor effects of RT alone according to fraction dose size and splitting schedules. Materials and Methods: 4T1 mouse breast cancer cells were implanted into the right and left sides of mammary gland fat pads of BALB/c mice, followed by irradiation with 6 Gy × 3, 8 Gy × 2, and 13 Gy × 1 fractions when the right-side tumors were palpable. Results: The different irradiation schedules produced similar antitumor effects in irradiated right-side tumors and unirradiated left-side tumors. However, 8 Gy × 2 and 13 Gy × 1 fractions exhibited better antimetastatic potential than that from irradiation using 6 Gy × 3 fractions. Furthermore, 8 Gy × 2 and 13 Gy × 1 fractions produced higher expressions of HMGB1 and lower expressions of the proinflammatory cytokines, IFN-γ, TNF-α, IL-6, and IL-1β, from the irradiated tumor tissues. Conclusions: These findings suggest that 8 Gy × 2 and 13 Gy × 1 fractions can provide better systemic antitumor effects than 6 Gy × 3 fractions. The authors hope these results provide clues to optimize RT dose regimens to make the abscopal effect clinically more relevant in future combination treatments.

Notch3 Knockout Suppresses Mouse Mammary Gland Development and Inhibits the Proliferation of 4T1 Murine Mammary Carcinoma Cells via CCL2/CCR4 Axis.

Our previous study found that Notch3 knockout mice exhibit defects in mammary gland development. To elucidate the underlying mechanism, tissue samples were subjected to RNA-seq, GO, and KEGG enrichment analyses and qRT-PCR validation. Of enriched pathways, chemokine signaling pathway and cytokine–cytokine receptor interaction were noticed in both Notch3wt/wt/Notch3wt/– and Notch3wt/wt/Notch3–/– mice, in which the expression of chemokine ligand 2 (CCL2) was sharply reduced in Notch3wt/– and Notch3–/– mammary gland tissues. The Mouse ENCODE transcriptome data reveal that the mammary gland fat pad exhibits a high CCL2, CCR2, and CCR4 expression, indicating that these molecules play important roles during mammary gland development. Specifically, defective mammary glands in Notch3 knockout mice could be partially rescued by CCL2 overexpression lentivirus through intraductal injection. An in vitro study showed that CCL2 overexpression promoted the proliferation, migration, and cancerous acinar formation of 4T1 cells, which could rescue the defective migration of 4T1 cells caused by Notch3 knockdown. We also found that Notch3 transcriptionally regulated the expression of CCL2 in a classical pattern. Our findings illustrated that Notch3-regulating CCL2/CCR4 axis should be an important signaling pathway for mammary gland development and should be a candidate target for breast cancer therapy.

Adipocyte-Derived Exosomes Increase the Growth of Breast Tumors in Obese Mice

Abstract Objectives 1) We assessed whether the secretion and cargos of exosomes are distinct in obese and lean mice. 2) We assessed whether adipocyte-derived exosomes increase the size of tumors of the mammary gland in mice. Methods Female tamoxifen-inducible adipose-specific TSG101 conditional knockout mice (TSG KO) were initiated on an AIN-93G-based 50% high fat (HFD) or 15.8% low fat (LFD) at age 3 weeks and continued on these diets for 10 weeks. At age 4 weeks, mice were injected with tamoxifen or vehicle (TSG+), followed by the injection of 1.8*106 murine breast adenocarcinoma E0771 cells into the mammary gland fat pad 10 days after tamoxifen injection. Loss and gain of TSG101 and Cre, respectively, in inguinal subcutaneous adipose tissue were confirmed by RT-qPCR. Tumors were excised and their size was assessed using a caliper and by weight. H&amp;E staining was used to assess the tumor type. Exosomes were isolated from plasma by an ExoQuick Plasma Prep kit, authenticated by western blot analysis and counted using a Nanosight tracker. The statistical analysis was performed by one-way ANOVA and Least Significant Differences posthoc test using a significance level of P &amp;lt; 0.05. Results HFD led to an increase in breast tumor size, which was diminished in adipose-specific TSG101 KO fed HFD: The size of mammary tumors in TSG+ fed HFD increased by 198% compared to TSG+ fed LFD (P &amp;lt; 0.05), whereas tumor size decreased by 58% TSG101 KO fed HFD compared to TSG+ fed HFD (P &amp;lt; 0.05). HFD led to an increase in adipose-derived exosomes that was reduced by TSG101 KO: The expression of ASC-1, a marker for adipose-derived exosomes in plasma exosomes increased by 58% in TSG+ fed HFD compared to TSG+ fed LFD, and decreased by 29% in TSG KO fed HFD compared to TSG+ fed HFD (P &amp;lt; 0.05). Plasma exosomes tested positive for exosome markers, CD63, CD81 and HSP70 and negative for a non-exosome marker, calnexin. H&amp;E staining suggested that the tumors observed in all mice were adenocarcinomas. Tumor numbers were not affected by TSG101 and diet. Conclusions HFD increases the growth of mammary tumors in mice and the diet effect depends on exosome biogenesis in adipose tissue. We speculate that adipose-derived exosomes promote tumor growth but not metastasis. Funding Sources NIFA, NIH, and USDA. J.Z. is a consultant for PureTech Health.

SAT-LB26 A Dual Role for IGF-1R in Mammary Tumorigenesis

The insulin-like growth factor type 1 receptor (IGF-1R) is now thought to have a dual function in breast cancer. Several studies have shown overexpression of the IGF-1R pathway results in increased tumor cell proliferation and survival. Recent loss-of-function models have shown decreased mammary tumorigenesis latency and increased metastasis. These recent studies correlate with analyses of human patient datasets identifying worse overall survival with low IGF-1R expression. Similarly, inhibition of IGF-1R in the clinic has had no affect or has led to worse outcomes supporting the hypothesis that the IGF-1R may have tumor suppressive properties. Our prior published studies revealed loss of IGF-1R function results in heightened tumor epithelial stress, which alters the tumor microenvironment to be permissive for metastasis. Therefore, we asked does the loss of IGF-1R inherently change the tumor epithelium or is it simply the alterations of the microenvironment that lead to a metastatic primary tumor? We first analyzed cell invasion of primary tumor epithelial cells from a mouse tumor model driven by the Wnt1 oncogene (MMTV-Wnt1) and with reduced IGF-1R signaling by expression of a dominant-negative transgene (MMTV-dnIGF-1R). Epithelial cells from the MMTV-Wnt1/dnIGF-1R (bigenic) tumors invaded at the same rate as MMTV-Wnt1 tumor epithelial cells by tail vein injection suggesting invasive capacity is unchanged with reduced IGF-1R signaling. Interestingly, size of lung micrometastases from tail vein injected bigenic tumor epithelial cells was significantly reduced and the number also decreased over time in part due to a proliferative defect determined by immunostaining for pH3. Similarly, bigenic primary tumor epithelial cells injected into the mammary gland fat pad failed to form tumors suggesting alterations in cell adhesion. Consistent with this observation, E-cadherin gene and protein expression were decreased in the bigenic tumor epithelium compared to MMTV-Wnt1 tumors. In vitro analysis of cell adhesion in MMTV-Wnt1 primary epithelial cells resulted in both K8+ (luminal) and K14+ (basal) tumor epithelial cells adherence, while only K14+ cells from bigenic tumors adhered to collagen. Similarly, the lung micrometastases from tail vein injections exhibited predominantly K14+ cells. Analysis of MMTV-Wnt1 and bigenic primary tumors using single cell RNAseq revealed alterations in both stromal and epithelial populations that may contribute to bigenic primary tumor growth and metastasis. These data support the conclusion that inhibiting IGF-1R signaling results in both alterations to the tumor epithelium (partial EMT) and microenvironment that result in metastasis, but also, that the IGF-1R deficient metastatic cells need a niche or paracine signaling to proliferate.

Abstract P4-14-11: Prolonged survival of mice bearing HER2 positive breast cancer with a therapeutic cancer vaccine via intra-reconstructed abdominal flap administration

Abstract Background: Previously we reported a porous silicon micro-particle based cancer vaccine, which stimulated robust CD8+ T cell-dependent anti-tumor immunity in mice bearing HER2-positive breast cancer. In this study we evaluated the efficacy of the vaccine via single injection into reconstructed abdominal flap after primary tumor mastectomy. Method: To generate murine model of HER2 positive breast cancer murine, 1 × 105 TUBO/luciferase cells were injectedinto both mammary gland fat pad and left ventricle of each 6-8 weeks old female Balb/c mice. Primary and systemic metastatic tumor growth were monitored with a IVIS-200 imaging system. Four days after tumor inoculation, mammary gland fat pad with primary tumor was resected by mastectomy, and superficial inferior epigastric(SIE) vessels based abdominal flap was used for abdominal reconstruction. During the surgery, mice also received one of the three treatments below: 1) negative control without treatments; 2) single intra-flap vaccination of cancer vaccine; and 3) single injection of cancer vaccine via footpad injection. Therapeutic efficacy were monitored with IVIS system weekly. To analyze immune response, popliteal and inguinal lymph nodes were collected one day and seven days post vaccination, and activation of dendritic cells and T cells was evaluated with a flow cytometry. HER2 specific T cells in splenocytes which highly expressed interferon-gamma (IFNγ) were counted by ELISpot. CD3+ T cell infiltration in brain metastatic nodules were analyzed by immunohistochemical assay. Result: Prolonged survival of mice bearing HER2-positive breast cancer was achieved by both intra-flap and intra-dermal vaccination of cancer vaccine. Mice vaccinated via intra-flap injection extended median survival by 9 days (p=0.025), although not as long as intra-dermal treatment (15 days, p=0.034), comparing to control group. In lymph nodes, activated dendritic cells increased in both two vaccinated groups. Interestingly, intra-flap vaccination induced higher response in inguinal lymph node, which considered as the major draining lymph node of this route. Meanwhile, intra-dermal vaccination raised higher immune response in popliteal lymph node. In ELISpot assay, IFNγ secreted T cells were found abundant in splenocytes from both two vaccination groups. Furthermore, efficient infiltration of T cells inside the brain metastatic nodules was found in intra-dermal injection group, which provided potential protection from distal recurrence after mastectomy. Conclusion: Cancer vaccine administrated via intra-flap injection effectively stimulated systemic immune response, and slowed down tumor progression in murine HER2 positive breast cancer model. Citation Format: Xiaoling Liu, Junhua Mai, Chaoyang Meng, Wei Wei, Haifa Shen. Prolonged survival of mice bearing HER2 positive breast cancer with a therapeutic cancer vaccine via intra-reconstructed abdominal flap administration [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-14-11.

Effect of increasing amounts of milk replacer powder added to whole milk on postweaning performance, reproduction, glucose metabolism, and mammary fat pad in dairy heifers

The aim of this study was to evaluate the effects of increasing the total solids (TS) content of liquid feed, by adding increasing amounts of milk replacer powder to whole milk, on age at puberty and postweaning performance, glucose metabolism, and mammary fat pad of dairy heifers. Crossbreed Holstein × Gyr heifers (n = 58) were distributed into 4 different treatments during the preweaning period. Treatments consisted of liquid feeds with TS content of 12.5 (actual TS = 13.5 ± 0.53%; n = 15), 15.0 (actual TS = 16.1 ± 0.03%; n = 15), 17.5 (actual TS = 18.2 ± 0.14%; n = 13), or 20.0% (actual TS = 20.4 ± 0.24%; n = 15), which were provided up to 59 d old. From 60 to 89 d old, the animals received the same starter offered during the preweaning period in addition to corn silage ad libitum starting at 70 d old. From 90 to 104 d old, the animals were adapted to a total mixed ration. At 105 d old, the animals were distributed in 4 paddocks equipped with electronic feed and water bins and were fed the same total mixed ration ad libitum. A period of adaptation to the electronic feed bins occurred from 105 to 119 d old. Feed and water intake and body weight and body frame development were assessed until puberty. Mammary gland evaluations were performed monthly by ultrasonography from 120 d of age until puberty onset. Puberty onset was determined as plasma progesterone concentration greater than 1 ng/mL in 2 consecutive samples collected 7 d apart. The date of puberty onset was recorded as the collection day of the first of these samples. A glucose tolerance test was performed at 280 d of age. The increased TS content of the liquid feed fed during the preweaning period did not affect dry matter intake, performance, age at puberty, glucose metabolism, or mammary gland fat pad at later stages of rearing process. Conversely, hip width and heart girth increased linearly up to 150 and 240 d of age, respectively, as a result of the increased TS concentration of the liquid feed during the preweaning period.

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpincpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpincpdm mammary epithelial cells transplanted invivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpincpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpincpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation invitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

Abstract P1-07-24: Modeling breast cancer metastasis in the mouse via measurement of circulating tumor cells

Abstract Objectives: Advances in the detection of circulating tumor cells (CTC) in the blood of metastatic breast cancer patients have indicated that patients with greater than 5 CTC/7.5mL of blood have poorer prognosis; however, little is known about the biology of these cells and what characteristics are critical for breast tumor cell dissemination. We are developing and evaluating several mouse models of breast cancer to characterize important markers that distinguish CTCs from other cell types in the blood as well as to assess whether silencing of various metastasis supporting genes leads to reductions in CTC counts and formation of metastatic tumors. Methods: This study involves two separate approaches to characterize CTCs. The first involves xenograft models using human breast cancer cells injected into the fat pad of immunocompromised mice. These cells are tagged with both mCherry, for detection in blood, and luciferase, to track metastatic spread of the primary tumor cells to other sites via intravital imaging. Blood was collected from tumor bearing mice at end point, processed and stained for characterized CTC markers. The second approach involves endogenous mouse models to characterize CTC counts and relevant markers in a variety of metastatic and non-metastatic murine breast cancer models using negative depletion to remove contaminating normal and hematopoietic cells. In both approaches CTCs have been assessed using the Imagestream by Amnis. Results: Initial xenograft experiments with MDA-MB-231 cells injected into the mouse mammary gland fat pad revealed a low concentration of mCherry+/cytokeratin+/Hoechst+/CD45- cells in the blood of mice without confirmed metastatic dissemination. Pilot studies on endogenous tumor models have demonstrated the utility of our negative depletion approach to remove contaminating hematopoietic cells as well as the detection of a population of EpCAM+/Hoechst+/CD45- cells. Conclusions: Initial experiments have provided feasibility for detailed characterization of mouse CTCs, which will be presented. This work complements our study being carried out on characterizing CTCs in metastatic breast cancer patients. Increasing understanding of the biology of circulating tumor cells and relating their characteristics to those of both the primary and metastatic tumors may reveal mediators common and functionally important to all 3 tumor cell populations and may therefore serve as better targets for treatments that are effective against both the primary and metastatic tumors. Citation Format: Jennifer L Gorman, Michael Parsons, Eldad Zacksenhaus, Sean Egan, Martin Chang, Jim R Woodgett. Modeling breast cancer metastasis in the mouse via measurement of circulating tumor cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-24.

ZnT2 is a critical mediator of lysosomal-mediated cell death during early mammary gland involution.

Mammary gland involution is the most dramatic example of physiological cell death. It occurs through an initial phase of lysosomal-mediated cell death (LCD) followed by mitochondrial-mediated apoptosis. Zinc (Zn) activates both LCD and apoptosis in vitro. The Zn transporter ZnT2 imports Zn into vesicles and mitochondria and ZnT2-overexpression activates cell death in mammary epithelial cells (MECs). We tested the hypothesis that ZnT2-mediated Zn transport is critical for mammary gland involution in mice. Following weaning, ZnT2 abundance increased in lysosomes and mitochondria, which paralleled Zn accumulation in each of these organelles. Adenoviral expression of ZnT2 in lactating mouse mammary glands in vivo increased Zn in lysosomes and mitochondria and activated LCD and apoptosis, promoting a profound reduction in MECs and alveoli. Injection of TNFα, a potent activator of early involution, into the mammary gland fat pads of lactating mice increased ZnT2 and Zn in lysosomes and activated premature involution. Exposure of cultured MECs to TNFα redistributed ZnT2 to lysosomes and increased lysosomal Zn, which activated lysosomal swelling, cathepsin B release, and LCD. Our data implicate ZnT2 as a critical mediator of cell death during involution and importantly, that as an initial involution signal, TNFα redistributes ZnT2 to lysosomes to activate LCD.