Fat Pads Of Mice Research Articles

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

Human ESC-derived Expandable Hepatic Organoids Enable Therapeutic Liver Repopulation and Pathophysiological Modeling of Alcoholic Liver Injury

Introduction: Here, we report the generation of human ESC-derived, expandable hepatic organoids (hEHOs) enabling therapeutic liver repopulation and pathophysiological modeling of alcoholic liver injury. Methods: The culture system including a mix of A83-01, Forskolin, R-spondin 1, Wnt3a and EGF along with the extracellular matrix was used to generate hEHOs. The liver repopulation potential of hEHOs was assayed in FRG mouse model of inherited metabolic liver disorders. The tumorigenesis and lineage restriction of hEHOs were evaluated by transplanting the hEHOs into epididymal fat pads of NOD/SCID mice. To model alcoholic liver disease, a derivative model by incorporating human fetal liver mesenchymal cells (hFLMCs) into the hEHOs, referred to as hFLMC/hEHO was generated and treated with 100mM ethanol for 7days. Results: The hEHOs stably maintain phenotypic features of bipotential liver stem/progenitor cells that can differentiate into functional hepatocytes or cholangiocytes. The hEHOs can expand for 20 passages enabling large scale expansion to cell numbers requisite for industry or clinical programs. The hEHOs display remarkable repopulation capacity in injured livers of FRG mice following transplantation. If implanted into the epididymal fat pads of immune-deficient mice, they do not generate non-hepatic lineages and have no tendency to form teratomas. The hFLMC/hEHO can model alcoholic liver disease-associated pathophysiologic changes, including oxidative stress generation, steatosis, inflammatory mediators release and fibrosis, under ethanol treatment. Conclusion: The hEHOs have considerable potential to be a novel, ex vivo pathophysiological model for studying alcoholic liver disease as well as a promising cellular source for treating human liver diseases.

MicroRNA-941 regulates the proliferation of breast cancer cells by altering histone H3 Ser 10 phosphorylation

Breast cancer including triple negative breast cancer (TNBC) represents an important clinical challenge, as these tumours often develop resistance to conventional chemotherapeutics. MicroRNAs play a crucial role in cell-cycle regulation, differentiation, apoptosis, and migration. Herein, we performed Affymetrix Gene Chip miRNA 4.0 microarray and observed differential regulation of miRNAs (75 upregulated and 199 downregulated) in metastatic MDA-MB-231 cells as compared to immortalized human non-tumorigenic breast epithelial (MCF-10A) cells. MicroRNA-941 was significantly upregulated in MDA-MB-231 cells (almost nine-fold increase) in comparison to MCF-10A cells. Transfection of MiRNA-941 inhibitor significantly decreased the proliferation and migration of MDA-MB-231 cells by altering the expressions of p21, Cyclin D1, PP2B-B1, E-cadherin and MMP-13. Interestingly, we provide first evidence that inhibiting miR-941 prevents cell proliferation and phosphorylation of histone H3 at Ser10 residue. Xenograft model of breast cancer was developed by subcutaneous injection of MDA-MB-231 cells into the mammary fat pad of female athymic nude mice (Crl:NU-Foxn1nu). The tumours were allowed to grow to around 60 mm3, thereafter which we divided the animals into seven groups (n = 5). Notably, intratumoral injection of miR-941 inhibitor significantly abolished the tumour growth in MDA-MB-231 xenograft model. 5-Fluorouracil (10 mg/kg, i.p.) was used as positive control in our study. To the best of our knowledge, we report for the first time that targeting miR-941 improves the sensitivity of MDA-MB-231 cells to 5-fluorouracil. This can be of profound clinical significance, as it provides novel therapeutic approach for treating variety of cancers (overexpressing miRNA-941) in general and breast cancers in particular.

Why is cancer so common a disease in people yet so rare at a cellular level?

Cancers are common diseases in people and yet, on a cellular level, are quite rare. The vast majority of both sporadic, spontaneous cancers and inherited germline cancers arise in single foci from singly transformed cells despite the fact that, in the former, carcinogenic factors bathe fields of millions of potential target cells and, in the latter, the predisposing germline mutations are present in every cell of a given organ and, in fact, every cell of the body. Although the multi-hit theory of carcinogenesis has been invoked to explain such things as cancer latency, which is the period between cancer initiation and emergence and the cancer-aging relationship where an accumulation of “hits” over a period of time are necessary for cancer emergence, the multi-hit theory falls short in explaining the rareness of transformation at a cellular level. This is so because many cancers are not due to multiple hits, and even for those that are, it would be expected that many cells would be exposed to those factors inducing the hits. Although the tumor stem/progenitor cell compartmental theory of tumorigenesis characterizes a tumor compartment that is capable of self-renewal and multipotency, accounting for cancer relapses and recurrences, this compartmental theory alone cannot account for the rareness of initial transformation at a cellular level as the cancer stem/progenitor cell compartment is already transformed and considerable in size. This study advances a different and novel hypothesis that oncogenesis is regulated and ultimately determined by a cell of origin’s critical state of differentiation. Before and after this critical state of differentiation has been reached, target cells cannot transform and give rise to cancer even when they receive the necessary carcinogenic insults or have the requisite transforming tumor suppressor genes or oncogenes. As support for this hypothesis, the study cites preliminary evidence using oncogene-containing transgenic mice that develop mammary carcinomas, to derive tail vein fibroblasts converted to iPSCs which, when left undifferentiated, and injected into the cleared fat pads of non-transgenic background mice give rise to mammary gland ontogeny and mammary gland carcinogenesis. However, when first differentiated in vitro into multiply different non-mammary lineages prior to injection, they fail to do so. The hypothesis has widespread implications for chemopreventive strategies.

Abstract 1667: Comparison of drug responses using patient-derived xenograft (PDX) and patient-derived organoid (PDO) models from treatment-refractory breast cancer

Abstract Background: Patient-derived xenografts (PDX) and organoids (PDO) have become important translational model systems for cancer research, especially for drug responses prediction. Since PDX models have limitation as low success rate and takes long time, PDO is emerging as a new technique. Here, we compared the drug responses in PDX and PDO models using tissues obtained from treatment-refractory breast cancer patients. Methods: Tumor tissues from breast cancer patients were implanted into the mammary fat pads of immunodeficient mice. Tumor size of mice were measured 3 times a week using digital caliper. When xenograft tumors reached 200mm3 in size, drug treatment was started. Drugs selection was based on gene expression patterns, the presence of available drugs, and clinical treatment history. The organoids were established from PDX tumor pieces. Organoids were seeded and cultured in 96-well plates (2000 cells per well) for drugs testing. We treated with a single or combination drugs in PDX and PDO models. For interpretation of drug sensitivity results of PDO or PDX, we referred to IC50 database of 2D cell lines or results from references. Results: We compared the drugs response efficacy in five cases with paired PDX and PDO models; 1 hormone receptor (HR) positive+; HER2 negative-, 1 HR+;HER2+ and 3 TNBC (triple-negative breast cancer) subtypes. In HR+;HER2- case, drug test results between PDX and PDO partially matched in single treated group. In HR+;HER2+, the results of combination treated groups were partially matched in PDX and PDO model. The results for two TNBC samples matched in single or combination treated groups. Especially, tumor size or cell viability of one TNBC case showed significant differences between control and sorafenib/everolimus combination treated groups. The other case of TNBC type had partially matched in PDX and PDO model. We will analyze the consistency for the genomic profiles of tumors in patients, PDX, and PDO models. Conclusion: We have compared the various drugs responses through the successful establishment of PDO and PDX from the different breast cancer subtypes. Although the results are not perfectly matched, it showed that these models have potential to assist the chemotherapy strategies for each patient and predict outcome of treated patient's prognosis. In the future, we will be focused on explaining why the results of drug response between PDX and PDO were inconsistent. (This study was supported by National Cancer Center, Korea, 1710450, and 1810101) Citation Format: Jin-Sun Ryu, You-sun Noh, Bo-Ra Kim, Yun-Hee Kim, A-Ra Jeon, Sung Hoon Sim, In Hae Park, Eun Gyeong Lee, Eun Sook Lee, Keun Seok Lee, Sun-Young Kong. Comparison of drug responses using patient-derived xenograft (PDX) and patient-derived organoid (PDO) models from treatment-refractory breast cancer [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 1667.

Abstract LB-079: Microbubble mediated focused ultrasound therapy enhances the antitumor potency and durability of anti-PD-L1 checkpoint blockade

Abstract Checkpoint inhibitor therapies (CITs) are fundamentally impacting the treatment landscape of many cancer types. Unfortunately, only limited subsets of patients show durable complete responses (CRs). While considerable efforts are therefore being made to combine CITs with other therapies, the issue of increased toxicity associated with this approach remains a challenge. Physical methods for enhancing CITs are also being investigated and may have the advantage of not adding to toxicity. One such approach is therapeutic ultrasound, or focused ultrasound (FUS), which can act through a range of mechanisms and is known to be associated with immunological effects. A prominent form of FUS uses systemically injected ‘microbubbles' (MBs) which oscillate in response to ultrasound to enhance vascular permeability or elicit vascular damage. Here we investigate the use of FUS+MBs to enhance the effects of a clinically relevant combination anti-PD-L1 plus paclitaxel (PTX) in the treatment of triple negative breast cancer. Mammary carcinoma tumors (EMT6) were initiated in the mammary fat pads of immune competent Balb/c mice. At day 8 post implantation, experiments were conducted with 4 groups: sham, FUS+MBs, aPD-L1+PTX, FUS+MBs+aPD-L1+PTX. anti-PD-L1 (clone:6E11; 5 mg/kg; Genentech) was given i.p. every 3-4 days for a total of 10 doses. PTX was administered i.p. biweekly for 3 doses. FUS+MBs exposures consisted of a single treatment at pressures high enough to elicit vascular shutdown, as determined through histology. The results (Table 1) demonstrated that the addition of FUS+MB treatment profoundly enhances the effects of aPD-L1+PTX (p<0.02) and resulted in CR for 7/8 tumors. Re-challenge experiments in the contralateral fat pad of CR mice revealed either no re-growth or delayed growth, consistent with immune memory being engaged. Day 10 Tumor Volumes ± SEMDay 10 Tumor Volumes (1 way-ANOVA; post-hoc: Tukey's)Kaplan-Meir Survival Analysis (Log-rank test)Animals with complete response(Sham) vs. (PTX + aPD-L1 + FUS + MBs)793.6 ± 193.4 vs. 105.6 ± 16.8p=0.0087p<0.00010/7 vs. 7/8(PTX + aPD-L1) vs. (PTX + aPD-L1 + FUS + MBs)690.8 ± 217.2 vs. 105.6 ± 16.8p=0.039p=0.0222/6 vs. 7/8(FUS + MBs) vs. (PTX + aPD-L1 + FUS + MBs)255.0 ± 102.4 vs. 105.6 ± 16.8p=0.85p=0.135/9 vs. 7/8(Sham) vs. (FUS + MBs)793.6 ± 193.4 vs. 255.0 ± 102.4p=0.043p=0.0010/7 vs. 5/9(Sham) vs. (PTX + aPD-L1)793.6 ± 193.4 vs. 690.8 ± 217.2p=0.96p=0.0390/7 vs. 2/6(PTX + aPD-L1) vs. (FUS + MBs)690.8 ± 217.2 vs. 255.0 ± 102.4p=0.16p=0.342/6 vs. 5/9 As FUS is a rapidly growing treatment modality in oncology, these results may ultimately have significant implications for enhancing CITs without added toxicity. This approach warrants further investigation, including its potential impact on immune memory. Citation Format: David Goertz, William Cruz, Sharshi Bulner, Alex Wright, Robert Kerbel. Microbubble mediated focused ultrasound therapy enhances the antitumor potency and durability of anti-PD-L1 checkpoint blockade [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 LB-079.

Abstract 2645: Obesity promotes tumor growth, reduces breast tumor latency and correlates with neovascularization: Therapeutic and diagnostic implications

Abstract Obesity is associated with an increased risk of, and a poor prognosis for, postmenopausal (PM) breast cancer (BC). The goal of this study was to determine whether diet-induced obesity (DIO) promotes (i) shorter tumor latency, (ii) escape from tumor dormancy, and (iii) acceleration of tumor growth, angiogenesis and metastasis and to elucidate the underlying mechanism/s. Two different BC models, an orthotopic model (luciferase/MDA-MB-436 injected into the mammary fat pad (MFP) of SCID mice) and a transgenic model (doxycycline-driven conditional MMTV/TWNT/luciferase) were utilized. To model the effect of obesity on PM BC, mice were ovariectomized (OVX) before being placed on a DIO (obese) or normal chow (NC; lean) regimen. In both the orthotopic and transgenic BC models, obese mice had significantly higher tumor frequency, higher tumor volume, and lower overall survival compared to lean mice. Breast tumors in OVX/DIO mice had significantly higher microvessel density (MVD), SMA+ mature vessels and increased aggressive local invasion into the surrounding fat pad and muscle. OVX/DIO TWNT mice displayed a significantly shorter tumor latency period (7 vs. >24 wk, P=0.003) compared to OVX/NC mice. Levels of VEGF, IL-6, bFGF, Lcn-2 and MMP-9 were higher, whereas Tsp-1 levels were lower, in the sera/urine of DIO mice during tumor progression, suggesting that these circulating factors could serve as surrogate biomarkers of BC development. OVX/DIO tumors had increased expression of the angiogenic factors VEGF and bFGF and higher pVEGFR2. Systematic time-course analysis of MFP in the TWNT BC model indicated that DIO MFP achieved advanced tumor proliferative and angiogenic status at significantly earlier time points compared to NC MFP. DIO MFP also exhibited an increased incidence of extensive hyperplasia and carcinoma in situ at earlier time points, suggesting that obesity promotes tumor initiation as well as progression. Hyperplastic ducts and microscopic neoplastic lesions (≤1mm) in DIO MFP in the early phases of tumor development displayed increased Ki67 staining and were associated with significantly higher numbers of blood vessels. Collectively, our data suggest that PM obesity reduces breast tumor latency, promotes escape from tumor dormancy and initiates aggressive tumor growth via increased angiogenesis. Consistent with these findings, targeting angiogenesis via a small molecule inhibitor resulted in delayed tumor formation compared to vehicle-treated mice in the OVX/DIO TWNT BC model, suggesting that targeting angiogenesis may be a useful strategy in obesity-driven breast tumor initiation and progression. Ongoing studies in our laboratory are investigating the mechanism(s) by which an obesogenic microenvironment mediates these effects. [This work was supported by NIH RO1CA185530, the Karp Family Foundation and the Breast Cancer Research Foundation] Citation Format: Roopali Roy, Jiang Yang, Lauren Merritt, Justine Alluine, Lewis A. Chodosh, Marsha A. Moses. Obesity promotes tumor growth, reduces breast tumor latency and correlates with neovascularization: Therapeutic and diagnostic implications [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 2645.

Abstract 3971: The role of obesity-associated adipose tissue inflammation in breast cancer metastasis

Abstract Although obesity is a known risk factor for breast cancer incidence, metastasis, and mortality, the mechanisms underlying this link remain elusive. This study therefore aims to uncover the relationship between obesity-associated adipose tissue and breast cancer metastasis by identifying specific inflammatory mediators in the adipose secretome. It is well known that obesity induces hypoxic conditions in the adipose tissue, stimulating an inflammatory response that ultimately creates a favorable environment for cancer cell growth and survival. The link between obesity-associated adipose inflammation and cancer metastasis, however, remains poorly described. We have previously shown that injection of basal-like E0771's, and claudin-low metM-Wnt-Lung and metM-Wnt-Liver metastatic breast cancer cell lines in mice fed a diet-induced obesity (DIO, 60% kcal from fat) diet displayed larger tumors with increased metastatic potential compared to lean mice fed a control (10% kcal from fat) diet. When diets were supplemented with Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), the enhancing effects of DIO on tumor growth and metastasis were mitigated. Gene expression microarray analysis of non-tumor bearing mammary fat pads from these mice revealed increased mammary expression of inflammation-related genes in tissues from DIO relative to control mice. Moreover, gene expression analysis of the tumor microenvironment from these mice revealed a signature of DIO-induced immunosuppression that was normalized by Sulindac supplementation. To further investigate the relationship between obesity-associated inflammation and breast cancer metastasis, we cultured our metastatic metM-Wnt-Lung, and metM-Wnt-Liver, and claudin-low, non-metastatic M-Wnt breast cancer cell lines with conditioned media (FCM) from the mammary fat pads of mice fed control, DIO, or DIO+Sulindac diets. We then conducted phenotypic assays to characterize differences in cell growth, proliferation, and metastatic potential. After normalizing the FCM concentrations to fat pad mass, we saw increased cell viability and migration in cells treated with FCM from DIO relative to control mice. Luminex assays identified several pro-inflammatory factors associated with the pro-cancer effects of FCM from DIO versus control mice, and additional characterization studies utilizing high-throughput proteomic screening and immune-depletion approaches are on-going. Taken together, our findings suggest an important role for the adipose secretome, and specifically inflammatory mediators, in obesity driven breast cancer progression. Citation Format: Lydia K. Eisenbeis, Shannon B. McDonell, Laura A. Smith, Alyssa J. Cozzo, Michael F. Coleman, Stephen D. Hursting. The role of obesity-associated adipose tissue inflammation in breast cancer metastasis [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 3971.

Inhibition of the mitotic kinase PLK1 overcomes therapeutic resistance to BET inhibitors in triple negative breast cancer.

The inhibition of bromo- and extraterminal domains (BET) has shown an anti-proliferative effect in triple negative breast cancer (TNBC). In this article we explore mechanisms of resistance to BET inhibitors (BETi) in TNBC, with the aim of identifying novel ways to overcome such resistance. Two cellular models of acquired resistance to the BET inhibitor JQ1 were generated using a pulsed treatment strategy. MTT, colony formation, and cytometry assays revealed that BETi-resistant cells were particularly sensitive to PLK1 inhibition. Targeting of the latter reduced cell proliferation, especially in resistant cultures. Quantitative PCR analysis of a panel of mitotic kinases uncovered an increased expression of AURKA, TTK, and PLK1, confirmed by Western blot. Only pharmacological inhibition of PLK1 showed anti-proliferative activity on resistant cells, provoking G2/M arrest, increasing expression levels of cyclin B, pH3 and phosphorylation of Bcl-2 proteins, changes that were accompanied by induction of caspase-dependent apoptosis. JQ1-resistant cells orthotopically xenografted into the mammary fat pad of mice led to tumours that retained JQ1-resistance. Administration of the PLK1 inhibitor volasertib resulted in tumour regression. These findings open avenues to explore the future use of PLK1 inhibitors in the clinical setting of BETi-resistant patients.

144-OR: The Antirheumatic Drug Auranofin Improves the Metabolic Phenotype of Obesity

Obesity is associated with chronic low-grade inflammation, reflecting significant alterations in the composition of immune cell populations that reside in white adipose tissue (WAT). The ensuing pro-inflammatory environment likely impinges on the metabolic functions of adipocytes and may partially explain the insulin resistance that characterizes type 2 diabetes mellitus (T2DM). We recently demonstrated ectopic expression of the microRNA miR-30a in the subcutaneous fat pad of diabetic mice coupled improved insulin sensitivity and increased energy expenditure with decreased ectopic fat deposition in the liver and reduced WAT inflammation. We subsequently pursued the notion that pharmacological inhibitors of inflammation that exhibit a gene expression profile similar to ectopic miR-30a expression in WAT should represent new drugs for insulin resistance and T2DM. To this end, we used the Broad Connectivity Map to analyze a library of small molecules that induce mRNA profiles similar to that of exogenous miR-30a expression in WAT. Through this process, we nominated the rheumatoid arthritis compound auranofin. Treatment of obese mice with auranofin improved insulin sensitivity and glucose tolerance. Auranofin treatment also normalized other obesity-associated abnormalities, including hepatic steatosis, serum insulin and leptin. Mechanistically, a combination of proteomics and immunophenotyping established auranofin likely exerts antidiabetic activities by opposing low-grade inflammation in WAT and ectopic liver fat accumulation. These studies reveal important metabolic properties of anti-inflammatory treatments that may be re-purposed as therapies for insulin resistance and T2DM. Disclosure A. Cox: None. P.M. Masschelin: None. N. Chernis: None. P. Saha: None. Z. Lian: None. J.B. Felix: None. K. Kim: None. H. Wu: None. S.M. Hartig: None. Funding American Diabetes Association (1-18-IBS-105 to S.M.H.); National Institutes of Health (R01DK114356)

A Polyphenol-Rich Muscadine Grape Extract Inhibits HER + Breast Cancer and Protects Against Trastuzumab-Induced Cardiotoxicity

Abstract Objectives Human epidermal growth factor receptor 2 positive (HER2+) breast cancer (BC) is characterized by overexpression of the HER2 receptor. Although targeted HER2 drugs such as trastuzumab (TRZ) are effective for some patients, many patients experience resistance and cardiotoxicity, highlighting the need for additional therapies. The objective of this study was to determine the effects of a polyphenol-rich muscadine grape skin/seed extract (MGE) on TRZ-sensitive and –resistant HER2 + BC and TRZ cardiotoxicity. Methods Human HER2 + BC cells were treated with MGE and/or TRZ and proliferation was analyzed using the IncuCyte® ZOOM. Synergy was assessed using the Chou-Talalay method. Tumor growth and cardiotoxicity were measured in HER2 + xenografts treated with MGE and/or TRZ. TRZ-resistant JIMT-1 cells (2 × 106) were injected in the fourth mammary fat pad of female nude mice and, when tumors reached 50 mm3, mice were divided into four groups: control, MGE, TRZ, MGE + TRZ (n = 10/group, five week treatment). Cardiac parameters were measured using echocardiography and markers of proliferation/oxidative stress were measured by immunohistochemistry. Results The combination of MGE and TRZ inhibited proliferation to a greater extent than either agent alone and was synergistic in TRZ-sensitive SKBR3 cells at five dose combinations. MGE reduced TRZ-resistant JIMT-1 tumor volume by 35% (p ≤ 0.05); however MGE + TRZ inhibited tumor growth to a greater extent (62%) than either agent alone [combination vs. MGE (p ≤ 0.05); combination vs. TRZ (p ≤ 0.01)]. In tumors, MGE + TRZ reduced Ki-67 and activated AKT to a greater extent than MGE or TRZ alone [combination vs. MGE (p ≤ 0.05); combination vs. TRZ (p ≤ 0.0001)]. TRZ significantly reduced left ventricular ejection fraction and fractional shortening, which was prevented by MGE (p ≤ 0.01). Additionally, MGE significantly attenuated the 3-fold TRZ-induced increase in the oxidative stress marker 4-hydroxynonenal in the left ventricle (p ≤ 0.0001). Conclusions MGE inhibited the proliferation of HER2 + BC and was synergistic when combined with TRZ while protecting against TRZ cardiotoxicity. Thus, MGE may serve as an effective therapeutic either administered singly or in combination with TRZ for the treatment of HER2 + BC. Funding Sources Chronic Disease Research Fund.

Interleukin-31 Receptor α Is Required for Basal-Like Breast Cancer Progression.

Purpose: Interleukin-31 receptor α (IL31RA) usually mediates IL-31 induced inflammation and allergic diseases. However, the functional roles of IL-31/IL31RA signaling in basal-like breast cancer (BLBC) progression remain totally unclear.Methods: Tumorsphere formation, transwell, and wound healing assays were used to measure the BLBC progression. We implanted tumor cells in mammary fat pad and tail vein of nude mice to detect the growth and metastasis of BLBC cells. Luciferase and ChIP assays were employed to measure the transcriptional regulation. Western blot and real-time PCR assays as well as bio-informatics analyses were conducted to observe the expression of IL31RA.Results: We found that silencing of IL31RA suppresses the cancer stem cell-like properties, migration and invasion of BLBC cells in vitro as well as tumor growth and metastasis in vivo. Knockdown of IL31RA ameliorates IL-31-mediated pro-oncogenic functions. Overexpression of IL31RA in luminal breast cancer cells enhances the cancer stem cell-like properties and cell motility. Our data further identified IL31RA as a target gene of Twist/BRD4 transcription complex.Conclusion: Overall, these data indicate that IL31RA promotes basal-like breast cancer progression and metastasis, suggesting that targeting of IL-31/IL31RA axis might be beneficial to treatment of BLBC.

Simultaneous targeting of HER family pro-survival signaling with Pan-HER antibody mixture is highly effective in TNBC: a preclinical trial with PDXs

BackgroundThe human epidermal growth factor receptor (HER) family, notably EGFR, is overexpressed in most triple-negative breast cancer (TNBC) cases and provides cancer cells with compensatory signals that greatly contribute to the survival and development of resistance in response to therapy. This study investigated the effects of Pan-HER (Symphogen, Ballerup, Denmark), a novel mixture of six monoclonal antibodies directed against members of the HER family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs).MethodsFifteen low passage TNBC PDX tumor samples were transferred into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 100–200 mm3, mice were randomized (n ≥ 6 per group) and treated following three 1-week cycles consisting of three times/week intraperitoneal (IP) injection of either formulation buffer (vehicle control) or Pan-HER (50 mg/kg). At the end of treatment, tumors were collected for Western blot, RNA, and immunohistochemistry analyses.ResultsAll 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor downmodulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways AKT and ERK. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation.ConclusionsThe marked effect of Pan-HER in numerous HER family-dependent TNBC PDX models justifies further studies of Pan-HER in TNBC clinical trials as a potential therapeutic option.

SAT-588 Role of Adipose Tissue in Reproduction, Mammary Gland Development, Function and Cancer

To investigate the role of adipose tissue in the function of the mammary gland (MG) and reproductive system, we have examined lipodystrophic (LD) mice. LD mice of both sexes are sterile, but fertility was restored in both sexes with leptin injections. In addition, leptin was only needed for initial stages of pregnancy and not for parturition. A transplant of mouse embryonic fibroblasts (MEFs) led to the formation of an ectopic fat pad which also rescued the fertility in both sexes. However, pups born to rescued LD mothers died shortly after birth. We therefore examined the mammary glands of these mothers. MGs from LD mice were rudimentary and lacked terminal end buds. Leptin-injected and MEF rescued LD mice were able to become pregnant, showed normal pregnancy-associated glandular proliferation despite a smaller glandular area, were able to produce a small amount of milk that had grossly normal content of milk proteins and neutral lipids, but could not sustain pups to weaning. In order to separate the individual requirements for 1) adipokines such as leptin, 2) estradiol, and 3) epithelial-adipocyte interactions, we performed a series of experiments with both LD and ob (leptin-deficient) mice that received either estradiol or preadipocyte transplant. The resulting fat pad did not rescue the defect in MG development in LD mice. The defect also was not rescued with estradiol pellets. Ob/ob mice, like LD mice, lack leptin and estradiol, but retain adipose tissue. Ob mice have defective MG development. However, in striking contrast to LD mice, reconstitution of a WT fat pad in ob mice rescued the defect in MG development. Estradiol treatment did not rescue MG development in ob mice. Therefore direct interaction between mammary gland epithelia and adipocytes is a requirement for full invasion and expansion of the gland during puberty, but is not required for glandular proliferation during pregnancy and milk production.Given that excess adipose tissue is a risk factor for breast cancer we wanted to determine if breast cancer was affected by the absence of adipose tissue. LD mice were bred to MMTV-PyMT mice that develop spontaneous breast cancer. Remarkably, LD PyMT+ mice had accelerated growth of primary tumors compared to WT PyMT+ mice. Using our MEF transplant model future studies will be directed to understanding whether the accelerated breast cancer growth is due to loss of adipokines or altered epithelial-stromal interactions.

A Single Low Dose of Eribulin Regressed a Highly Aggressive Triple-negative Breast Cancer in a Patient-derived Orthotopic Xenograft Model.

In the present study, the breast cancer patient-derived orthotopic xenograft (PDOX) model was used to identify an effective drug for a highly aggressive triple negative breast cancer (TNBC). The TNBC tumor from a patient was implanted in the right 4th inguinal mammary fat pad of nude mice to establish a PDOX model. Three weeks later, 19 mice were randomized into the untreated-control group (n=10) and the eribulin treatment group (n=9, eribulin, 0.3 mg/kg, i.p., day 1). On day 8, eribulin significantly inhibited tumor volume compared to the control group (p<0.01). Eribulin regressed tumors in 3 mice (33.3%) and apparently eradicated them in 6 mice (66.7%). At day 14, tumor regrowth was observed in 2 mice of the eribulin group, which was undetectable on day 8. However, 44.4% (4 out of 9) of the mice in the eribulin group were tumor-free on day 14. A single low-dose eribulin was efficacious on a highly aggressive TNBC. The breast cancer PDOX model can be used to identify highly effective drugs for TNBC.

Dexamethasone Attenuates X-Ray-Induced Activation of the Autotaxin-Lysophosphatidate-Inflammatory Cycle in Breast Tissue and Subsequent Breast Fibrosis.

We recently showed that radiation-induced DNA damage in breast adipose tissue increases autotaxin secretion, production of lysophosphatidate (LPA) and expression of LPA1/2 receptors. We also established that dexamethasone decreases autotaxin production and LPA signaling in non-irradiated adipose tissue. In the present study, we showed that dexamethasone attenuated the radiation-induced increases in autotaxin activity and the concentrations of inflammatory mediators in cultured human adipose tissue. We also exposed a breast fat pad in mice to three daily 7.5 Gy fractions of X-rays. Dexamethasone attenuated radiation-induced increases in autotaxin activity in plasma and mammary adipose tissue and LPA1 receptor levels in adipose tissue after 48 h. DEX treatment during five daily fractions of 7.5 Gy attenuated fibrosis by ~70% in the mammary fat pad and underlying lungs at 7 weeks after radiotherapy. This was accompanied by decreases in CXCL2, active TGF-β1, CTGF and Nrf2 at 7 weeks in adipose tissue of dexamethasone-treated mice. Autotaxin was located at the sites of fibrosis in breast tissue and in the underlying lungs. Consequently, our work supports the premise that increased autotaxin production and lysophosphatidate signaling contribute to radiotherapy-induced breast fibrosis and that dexamethasone attenuated the development of fibrosis in part by blocking this process.

DETERMINING THE EFFECTS OF PRO‐ANGIOGENIC ELP‐VEGF THERAPY ON TUMOR GROWTH AND PROGRESSION

The members of the Vascular Endothelial Growth Factor (VEGF) family are endogenous growth factors with a central role in angiogenesis and endothelial cell health. We recently showed the therapeutic potential of renal VEGF delivery using a fusion protein of VEGF bound to the carrier protein Elastin‐like Polypeptide (ELP‐VEGF) designed in our laboratory, to promote angiogenesis in chronic kidney diseases. Indeed, we showed that ELP‐VEGF therapy improves renal vascular density, decreased renal fibrosis, and improved glomerular filtration rate (GFR) in swine models of chronic renovascular disease and chronic kidney disease at therapeutic doses of 0.1 – 1.0 mg/kg.A potential limitation of the use of ELP‐VEGF as a therapeutic is the possibility of promoting uninhibited off‐target angiogenic growth which may be undesirable in certain disease processes such as cancer. In order to determine whether ELP‐VEGF therapy may be harmful for a patient with an undiagnosed tumor, the effects of a single administration of escalating doses (0.1 m/kg, 1 mg/kg, 10 mg/kg, 10 times the highest therapeutic dose) of ELP‐VEGF on tumor size and vascular density were determined in a mouse xenograft model of breast cancer.Human triple‐negative breast cancer cells (MDA‐MB‐231) that stably express a luciferase reporter were implanted into the mammary fat pad of athymic nude mice. Tumors were monitored thrice weekly for 7 weeks with external caliper measurements (blind) and bioluminescence imaging to assess tumor growth. Body weights of the mice were also recorded 3 times per week. At the end of the 7‐week period, mice were fully perfused with heparinized saline and microfil. Tumors were removed and scanned using micro‐computed tomography (u‐CT), and 3D reconstructions of the tumor vascular network were used to blindly quantify tumor vascular density.Caliper measurements and bioluminescence imaging showed no significant difference in tumor size or growth rate amongst the groups. There were also no significance differences in mouse body weights throughout the study or final weights of the tumors (0.1–10mg/kg 1.12±0.39 g, 1.233±0.46 g, 1.247±0.52 g, respectively; p=0.8695). However, analysis of the renal vasculature by u‐CT revealed a significant increase in total microvascular density after a single injection of ELP‐VEGF at 1mg/kg (78.88 ± 34.34 vessels/mm2; p = 0.0489 vs. controls) and 10mg/kg (97.17 ± 48.65 vessels/mm2; p = 0.0012 vs. controls). In conclusion, we determined that one‐time injection of ELP‐VEGF had no toxic effects on body weights and did not influence tumor growth rate, but it did significantly and dose‐dependently increase tumor vasculature density in a breast cancer xenograft model.Support or Funding InformationR01HL095638 and IPA34170267Figure 2. Analysis and results from ELP‐VEGF‐A administration to a rodent model of breast cancer. Results include body weight (a), tumor caliper measurements (b) tumor bioluminescence (c), final tumor mass(d).Figure 1Figure 3. Vasculature of each tumor was visualized using uCT scanner after microfi fixation. Blood vessels were divided and quantified by diameter between different ranges (a). Kidneys from these mice were also perfused with microfil, scanned through uCT scanner, and quantified by vessel diameter range (b).Figure 2

A Collagen Based Cryogel Bioscaffold that Generates Oxygen for Islet Transplantation.

The aim of this work was to develop, characterize and test a novel 3D bioscaffold matrix which can accommodate pancreatic islets and provide them with a continuous, controlled and steady source of oxygen to prevent hypoxia-induced damage following transplantation. Hence, we made a collagen based cryogel bioscaffold which incorporated calcium peroxide (CPO) into its matrix. The optimal concentration of CPO integrated into bioscaffolds was 0.25wt.% and this generated oxygen at 0.21±0.02mM/day (day 1), 0.19±0.01mM/day (day 6), 0.13±0.03mM/day (day 14), and 0.14±0.02mM/day (day 21). Accordingly, islets seeded into cryogel-CPO bioscaffolds had a significantly higher viability and function compared to islets seeded into cryogel alone bioscaffolds or islets cultured alone on traditional cell culture plates; these findings were supported by data from quantitative computational modelling. When syngeneic islets were transplanted into the epididymal fat pad (EFP) of diabetic mice, our cryogel-0.25wt.%CPO bioscaffold improved islet function with diabetic animals re-establishing glycemic control. Mice transplanted with cryogel-0.25wt.%CPO bioscaffolds showed faster responses to intraperitoneal glucose injections and had a higher level of insulin content in their EFP compared to those transplanted with islets alone (P<0.05). Biodegradability studies predicted that our cryogel-CPO bioscaffolds will have long-lasting biostability for approximately 5 years (biodegradation rate: 16.00±0.65%/year). Long term implantation studies (i.e. 6 months) showed that our cryogel-CPO bioscaffold is biocompatible and integrated into the surrounding fat tissue with minimal adverse tissue reaction; this was further supported by no change in blood parameters (i.e. electrolyte, metabolic, chemistry and liver panels). Our novel oxygen-generating bioscaffold (i.e. cryogel-0.25wt.%CPO) therefore provides a biostable and biocompatible 3D microenvironment for islets which can facilitate islet survival and function at extra-hepatic sites of transplantation.

The lactate receptor GPR81 promotes breast cancer growth via a paracrine mechanism involving antigen-presenting cells in the tumor microenvironment

GPR81 is a G-protein-coupled receptor for lactate, which is upregulated in breast cancer and plays an autocrine role to promote tumor growth by tumor cell-derived lactate. Here we asked whether lactate has any paracrine role via activation of GPR81 in cells present in tumor microenvironment to help tumor growth. First, we showed that deletion of Gpr81 suppresses breast cancer growth in a constitutive breast cancer mouse model (MMTV-PyMT-Tg). We then used a syngeneic transplant model by monitoring tumor growth from a mouse breast cancer cell line (AT-3, Gpr81-negative) implanted in mammary fat pad of wild-type mice and Gpr81-null mice. Tumor growth was suppressed in Gpr81-null mice compared with wild-type mice. There were more tumor-infiltrating T cells and MHCIIhi-immune cells in tumors from Gpr81-null mice compared with tumors from wild-type mice. RNA-seq analysis of tumors indicated involvement of immune cells and antigen presentation in Gpr81-dependent tumor growth. Antigen-presenting dendritic cells expressed Gpr81 and activation of this receptor by lactate suppressed cell-surface presentation of MHCII. Activation of Gpr81 in dendritic cells was associated with decreased cAMP, IL-6 and IL-12. These findings suggest that tumor cell-derived lactate activates GPR81 in dendritic cells and prevents presentation of tumor-specific antigens to other immune cells. This paracrine mechanism is complementary to the recently discovered autocrine mechanism in which lactate induces PD-L1 in tumor cells via activation of GPR81 in tumor cells, thus providing an effective means for tumor cells to evade immune system. As such, blockade of GPR81 signaling could boost cancer immunotherapy.