Mouse Pad Research Articles

Abstract 1525: Vortex technology for label-free enrichment of CTC from mouse xenograft models

Abstract Background Non-invasive liquid biopsies, such as CTCs, have been of growing interest due to their potential use in cancer detection, prognosis, and monitoring of therapeutic resistance [1]. Beyond enumeration, characterization of CTCs could help guide treatment selection and the development of targeted cancer therapy. Here, we show that Vortex technology can be used successfully for the size-based capture of CTCs in a preclinical mouse model of breast cancer. Method To establish that human epithelial cancer cells can be reliably detected in small volumes of mice blood, 50-100 MDA-MB231 and MCF7 breast cancer cells were spiked in 500 μL mice blood, diluted 20 fold, and processed through Vortex chip [2]. Recovered cells were immunostained (CK, CD45, DAPI), and enumerated. For breast cancer xenograft model, 8×106 MDA-MB-231-fLuc/GFP cells were implanted orthotopically into the mammary fat pad of NOD-SCID Gamma mice (n = 35). Tumors were measured in 2 dimensions 3 times/week and tumor volume calculated. Blood from cardiac puncture (500 μl) and lateral saphenous vein (100 μl) was collected starting 1 week post implantation, diluted 40X and processed. Mice were euthanized, organs harvested, formalin fixed, paraffin embedded and H&E stained. Results Spiking of MCF7 cells in mouse blood resulted in a capture efficiency of 56.9±13% and a purity of 80.1± 9% (n = 3), with the flow-through from each cycle being re-processed for a total of 4 cycles. Recycling the flow-through led to capture efficiency of 36.7%, 23.4%, 5.2% and 7.9% during cycles 1, 2, 3 and 4 respectively. Similar results were obtained for MDA-MB-231, with a capture efficiency of 54.4% and a purity of 41.3%. In an orthotopic tumor xenograft model of breast cancer, CTCs were successfully recovered from cardiac puncture as early as day 7. CTC counts, ranging from 0.4-3649 CTCs/100 μl, increased over time and correlated with tumor burden. CTC clusters were also captured from day 7 (6 clusters/100 μl; frequency 1/3), with number and frequency increasing over time up to 147-485 clusters/100 μl by day 42. No CTC were recovered from lateral saphenous vein blood until day 28 post implantation, and their number remained low (mean 2.15±0.65). Microscopic metastases were evident in lung of all mice starting at day 28 and in liver of all mice starting at day 35. Conclusion CTCs were isolated in a label-free manner from mice blood with both high capture efficiency and purity. In a preclinical model of metastatic breast cancer, CTC counts correlated well with primary tumor volume and metastases occurrence. Thus the Vortex chip appears to be well suited for the enrichment of CTCs from murine xenograft models. Future works will focus on mice implanted with patient derived xenograft (PDX) and their therapeutic response. Information gathered from these studies should facilitate discovery of new therapeutic targets and the development of personalized medicine. [1] Ignatiadis et al. Clin Cancer Res 2015. [2] Sollier et al. Lab Chip 2014.[K.H. and M.T. contributed equally to this work.] Citation Format: Kyra Heirich, Melanie M. Triboulet, Corinne M. Renier, Vishnu C. Ramani, Elodie Sollier, Stefanie S. Jeffrey. Vortex technology for label-free enrichment of CTC from mouse xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1525.

Abstract 1242: Combination of p21-activated kinase 1 (PAK1) inhibitor FRAX1036 and Aurora-A inhibitor alisertib is effective in hormone receptor-positive breast cancer

Abstract Estrogen receptor alpha (ERα) signaling plays a critical role in the development and progression of hormone receptor-positive (HR+) breast cancer. One mechanism of tumor progression of HR+ breast cancer is phosphorylation of ERα by non-receptor protein kinases, leading to ligand-independent activation of ERα. Mitotic kinases Aurora A (AURKA) and p21-activated kinase 1 (PAK1) are frequently overexpressed in breast cancer and can phosphorylate ERα on serine 305, increasing the ability of ERα to induce transcription of target genes implicated in tumorigenesis. In addition, PAK1 phosphorylates and activates AURKA and its functional partners, suggesting possible synergistic activity in tumors overexpressing both kinases. We tested the hypothesis that combined inhibition of AURKA and PAK1 would synergistically suppress tumor growth of HR+ breast cancer cells in vitro and in vivo. Treatment of T47D HR+ breast cancer cell line with the PAK1 inhibitor FRAX1036, combined with AURKA inhibitor alisertib, improved cell killing in comparison to single agents. FRAX1036 and alisertib were then evaluated in an orthotopic xenograft model using BT474 HR+ breast cancer cell line. Ten million BT474 cells were implanted into the mammary fat pads of 6-week old NSG mice simultaneously with implantation of estrogen pellets to facilitate growth of xenografts. Mice received 21 days of treatment with FRAX1036 20mg/kg daily and alisertib 10 or 15 mg/kg BID, alone or in combination. We observed exceptional inhibition of tumor growth in the combination treatment groups. Combination of FRAX1036 20mg/kg daily with alisertib 15mg/kg BID was the most effective, with partial or near-complete responses that continued through 21 days of treatment. This combination resulted in significantly better inhibition of tumor growth than alisertib 15mg/kg BID alone or FRAX1036 20mg/kg daily alone (p = 0.003 and p = 1.29e-6, respectively). Similarly, combination treatment of FRAX1036 20mg/kg daily with alisertib 10mg/kg BID was significantly better then alisertib alone or FRAX1036 alone in the corresponding doses (p = 0.002 and p = 3.52e-7, respectively). Analysis of H&E stained sections and Ki-67 proliferation index of BT474 xenograft tumors indicated a highly significant decrease in viable tumor cells and replacement by stroma after combination treatment compared to treatment with single agents or with control. Caspase-3 IHC analysis showed significantly greater proportion of apoptotic cells per tumor area in the group of mice treated with FRAX1036 and Alisertib 15mg/kg BID combination comparing to control (p = 0.008). We conclude that combining FRAX1036 and alisertib is a novel and promising approach in HR+ breast cancer. Citation Format: Elena Shagisultanova, Vladislav Korobeynikov, Anna Nikonova, Jonathan Chernoff, Virginia Borges, Erica Golemis. Combination of p21-activated kinase 1 (PAK1) inhibitor FRAX1036 and Aurora-A inhibitor alisertib is effective in hormone receptor-positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1242.

Abstract 2024: BP1 homeoprotein induces tumor cell growth and estrogen-independent tumorigenesis in mice

Abstract BACKGROUND. BP1 is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 overexpression increases cell survival, aggressiveness and metastasis in breast cancer cells. BP1 protein is expressed in 80% of invasive ductal breast tumors, including 100% of ER negative (ER-) tumors and 73% of ER positive (ER+) tumors. Therefore we hypothesize that BP1 may regulate ER in breast cancer. A mouse model was developed to test the regulatory effects of BP1 in ER expression. MATERIALS AND METHODS. MCF-7 breast cancer cells transfected with BP1 or vector control were njected into the fat pads of nude mice. Regulation of ER by BP1 was studied using ChIP assays; EMSA assays were used to test binding of BP1 to ER DNA. Tamoxifen assays were used to evaluate the effects of BP1 overexpression on tamoxifen resistance. RESULTS. MCF-7 cells overexpressing BP1 or control cells containing an empty vector were injected into nude mice. Approximately half of the estrogen-supplemented mice developed tumors. Although MCF-7 cells require exogenous estrogen to form tumors in nude mice, approximately 20% of the mice implanted with cells overexpressing BP1 were able to form tumors in the absence of added estrogen. To understand the molecular mechanisms of this observation, we carried out in vitro experiments. Our data suggest that BP1 regulates ER both directly and indirectly: (1) Direct: ChIP assays and EMSA assays showed that BP1 binds to the first intron of the ER alpha gene and transcriptionally activates it, leading to increased ER protein. (2) Indirect: regulation of ER also occurs indirectly by BP1 upregulation of p300, an activator of ER, and also by repression of BRCA1 by BP1; BRCA1 destabilizes ER protein. Furthermore, MCF-7 cells overexpressing BP1 are more resistant to growth in 3uM tamoxifen compared with empty vector containing cells. CONCLUSIONS. BP1 overexpression in MCF-7 cells injected into mice not only increases tumor size but also promotes estrogen-independent tumorigenesis, and leads to resistance to tamoxifen. Therefore, BP1 could serve as a novel therapeutic target for ER+ breast cancer. Citation Format: Sidney W. Fu, Saurabh Kirolikar, Erika Ginsburg, Arnold Schwartz, Samuel Simmens, Patricia E. Berg. BP1 homeoprotein induces tumor cell growth and estrogen-independent tumorigenesis in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2024.

Abstract 1656: Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation

Abstract Breast cancer is the most common malignancy diagnosed among women worldwide and represents a heterogeneous group of subtypes. Radiation therapy is an important component of multimodal treatment for women with breast cancer. However, little is known about radiation response among these subtypes. Recent clinical data suggests that distinct patterns of response to systemic therapy may exist in association with each phenotype. Therefore, understanding the intrinsic variation of radiation response in breast cancer subtypes is important in prescribing individualized radiotherapy that maximizes the therapeutic ratio. Our previous studies have identified marked changes in gene expression in breast patient tumors and breast tumor cell lines in response to radiation. Among candidate genes, FAS was consistently and significantly induced after radiation in luminal breast tumors and cell lines. Modulation of FAS in basal breast cancer cell lines favorably impacted radiation response. In this study, we further investigated the role of FAS modulation and radiation response in a mouse model of breast cancer. MCF7 (luminal), HCC1954 (HER2+) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of nude mice. These mice were then treated with different doses of radiation. Our results showed that radiation treatment inhibited MCF7, and to a less extent HCC1954 tumor growth in a dose-dependent manner, while SUM159 tumors were resistant to radiation. The estimated TCD50 value was 19.30 Gy for MCF7 and 44.88 Gy for SUM159. FAS was induced in MCF7 tumors after radiation but showed no change in SUM159 tumors. To investigate whether FAS modulation can affect radiation response, we silenced FAS in MCF7 tumors and activated FAS in SUM159 tumors. We found that silencing of FAS did not negatively impact radiation response in MCF7 tumors, possibly due to compensation by other apoptotic pathways. On the other hand, FAS activating antibody in combination with radiation delayed SUM159 and HCC1954 tumor growth (p = NS). In conclusion, these results suggest that there is intrinsic variation in radiation response among breast cancer subtypes. FAS concurrent with radiation slows tumor growth in the radiation resistant subtypes, but the effect was not significant. Alternative subtype-specific modulators of radiation response are under investigation. Citation Format: Chen-Ting Lee, Yingchun Zhou, Kingshuk R. Choudhury, Sharareh Siamakpour Reihani, Mark W. Dewhirst, Janet K. Horton. Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1656.

Abstract 1410: PI3K inhibitors reduce breast cancer tumor growth and enhance anti-tumor leukocyte activity

Abstract Background: PI3K inhibitors have shown anti-tumor activity in breast cancer and are currently being investigated in multiple clinical trials. Despite the number of trials studying treatment efficacy, tolerability, and survival, little attention has been given to how these agents affect the anti-tumor capacity of leukocytes. However, one potential reason for diminished success of these therapies could be that systemic inhibition of this kinase results in a loss of ‘entrainment’ of leukocytes such that they fail to appropriately kill tumor cells. Therefore, the goal of this research was to evaluate the effects of PI3K inhibitors on leukocyte function in breast cancer. Methods: Blood samples were obtained from patients enrolled in the VICCBRE1320 clinical trial aimed to evaluate GDC0032 and docetaxel. Entrainment properties of circulating leukocyte cells were compared from Day 0 to on treatment samples for multiple cycles of therapy. Blood samples were drawn before administration of the treatment. Peripheral blood leukocytes were isolated and analyzed by FACS. Neutrophils as well as CD4+/8+ cells were also isolated from these blood samples to measure their ability to kill breast cancer tumor cells in vitro. We also examined the effects of PI3K inhibitors on human breast cancer tumors and leukocytes using humanized mouse models. For these models, tumors were implanted into the mammary fat pad of NSG mice engrafted with the patient's CD34+ hematopoietic stem cells and infused with the patient's in vitro expanded CD4+/8+ T cells. Results: In two of the clinical trial patients studied we observed a decrease CD4+ T cells and DCs over the first two cycles of therapy (p<0.007). We also saw a drop in B, NK, Treg cells in one patient (p = 0.004). No changes in circulating neutrophils or macrophages have been detected. We found that neutrophils as well as CD4+/8+ cells isolated from the blood of these patients are able to kill ER+ MCF7 or Triple-Negative MDA-MB-231 tumor cells in vitro (p<0.038). Throughout the treatment period, leukocytes from patients receiving PI3K inhibitor are able to kill tumor cells regardless of the breast cancer subtype or PI3K mutation status. When we treated humanized mice bearing patient derived xenografts of triple negative breast tumors with BKM120, we observed a 20% decrease in CD4+ cells and an increase in Th1 phenotype among tumor infiltrating T cells. BKM120 also resulted in a decrease in tumor growth compared to vehicle (p<0.01). Conclusions & Future Directions: Our findings suggest that pan-Class I PI3K inhibitors possess anti-tumor activity, but are also capable of enhancing anti-tumor immune cell function. The latter could improve treatment outcomes as well as prevent metastasis. Hence, our preliminary data demonstrate that PI3K inhibitors can serve as effective therapies in breast cancer regimens to reduce tumor growth and progression. As part of a combination, these agents may also effectively treat metastatic patients. Citation Format: Nicole Lavender, Jinming Yang, Jiqing Sai, Sergey Novitskiy, Linda Horton, Andrew Johnson, Vandana Abramson, Ingrid Mayer, Ingrid Meszoely, Mark Kelley, Ann Richmond. PI3K inhibitors reduce breast cancer tumor growth and enhance anti-tumor leukocyte activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1410.

Abstract 3308: Bisphenol A treatment induces hyperplasia in primary and stem cell-generated mammary glands from pregnant mice

Abstract Breast cancer is a commonly diagnosed cancer during pregnancy. However, how endogenous and exogenous endocrine factors may contribute to the development of pregnancy-associated mammary tumorigenesis is not clear. There is growing evidence that mammary stem cells (MaSCs) may initiate neoplastic transformation when dysregulated in mouse models. We investigated the effect of the environmental endocrine disruptor bisphenol A (BPA) on mouse mammary gland morphology, epithelial cell composition, pre-neoplastic lesions, and the regenerative function of MaSCs. Pregnant FVB mice with GFP transgene on Day E8.5 were implanted with osmotic pumps that constantly release BPA at 0, 25 or 250 ng/kg/day for 28 days and the mice were euthanized one month after weaning. In agreement with the literature, we observed an abnormality of the morphology of the mammary gland after BPA treatment characterized by higher duct density and abnormal secondary and tertiary branching. Quantification of percent hyperplastic mammary ducts in H&E-stained tissue slides revealed a significant increase of ducts with hyperplastic lesions after BPA treatment, particularly with the low dose. To investigate the effects of BPA treatment on MaSCs, we used enzyme digestion to isolate the CD24hi/CD49f+ luminal epithelial cells (also termed as colony forming cell or CFC) and the CD24+/CD49fhi basal epithelial cells (also termed as mammary repopulating unit or MRU) from mammary gland tissues by FACS and found no significant difference in percent of luminal or basal cell population after BPA treatment. Because the basal cells are enriched with MaSCs that can form mammospheres in suspension culture and subsequently form solid 3D organoids when cultured in Matrigel, we transplanted the solid 3D organoids into cleared mammary fat pads of syngeneic FVB mice and immune-compromised nude mice to examine how BPA treatment might alter MaSC function. Significantly, similar to the results from the primary mammary glands, the regenerated mammary glands by MaSCs from mice treated with the low dose of BPA showed increased duct density, secondary and tertiary branching, and a significantly greater number of hyperplastic lesions. Taken together, our study demonstrated that BPA exposure at very low dose could induce pre-neoplastic lesions in the mammary gland of pregnant mice, apparently by directly targeting MaSCs, and implicates BPA as an exogenous endocrine factor that may promote pregnancy-associated mammary tumorigenesis. Citation Format: Hakim Bouamar, Fuchuang Zhang, Xiang Gu, Qiaoxiang Dong, Lu-Zhe Sun. Bisphenol A treatment induces hyperplasia in primary and stem cell-generated mammary glands from pregnant mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3308.

Abstract 392: Combined targeting of MET and PI3K improves efficacy in breast cancer models with concurrent MET/PI3K aberrations

Abstract Introduction: Detailed understanding of the genetic abnormalities that drive subsets of cancer has led to the development of highly specific inhibitors targeting key oncogenic pathways. Growing evidence implicates aberrations of both mesenchymal epithelial transition receptor (MET) and its key downstream phosphatidylinositol 3-kinase (PI3K) signaling with poorer prognosis in breast cancer. Co-targeting MET/PI3K and identifying mechanisms of response and resistance could ultimately inform a novel therapeutic strategy for breast cancer treatment. Based on these finding, we hypothesized that concurrent aberrations in PI3K and MET will render breast cancers resistant to therapies targeting each pathway, and that combination therapy targeting the PI3K and MET pathway will improve the efficacy by preventing the acquisition of resistance. Methods: We established cell models that stably express human wild type (WT) MET or MET-T1010I, a functional germline single nucleotide polymorphism, or mutant PI3K-H1047R alone, or variable concurrent aberrations in PI3K and MET using MCF-10A, a non-transformed mammary epithelial cell line, and HCC1954, a breast cancer cell line with an endogenous PI3KCA-H1047R mutation. Cell behaviors, including survival, invasion and response to MET one arm monoclonal antibody onartuzumab (MetMAb, Genentech Inc) and/or pan class I PI3K inhibitor GDC 941 (pictilisib, Genentech Inc), were detected. HCC1954 expressing WT MET or MET-T1010I along with PI3K-H1047R were transplanted into the mammary fat pad of hepatocyte growth factor-transgenic mice on a severe combined immunodeficiency background. Statistical analyses were carried out using the ANOVA and the Student t test. Result: Expression WT MET or MET-T1010I act coordinately with PI3K-H1047R to increase colony formation (P < 0.01, respectively). Concurrent MET-T1010I, but not WT MET, acts coordinately with PI3K-H1047R to increase cell invasion (P < 0.0001). Expression MET-T1010I or WT MET induced cells resistance to GDC941, with MET-T1010I stronger than WT MET. Combined targeting MET with onartuzumab and targeting PI3K with GDC941 significantly inhibited cell growth in matrigel, dose dependently than each monotherapy (P < 0.0001, respectively). Invasion assay showed similar pattern. Combined onartuzumab and GDC941 significantly inhibited invasion, dose dependently (P < 0.001). The effects were confirmed using PI3K/mTOR inhibitor, GDC980. Consistently, combination of onartuzumab/GDC941 significantly inhibited tumor growth (P < 0.01 versus GDC941; P < 0.0001 versus onartuzumab alone). Conclusions: Concurrent aberrations of MET and PI3K render breast cancer cells resistant to therapies blocking either pathway. Targeting both MET and PI3K increases inhibitory efficacy. Citation Format: Shuying Liu, Naoto T. Ueno, Bailiang Wang, Mihai Gagea, Prahlad T. Ram, Mark Merchant, John Mendelsohn, Debasish Tripathy, Gordon B. Mills, Ana M. Gonzalez-Angulo. Combined targeting of MET and PI3K improves efficacy in breast cancer models with concurrent MET/PI3K aberrations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 392.

Abstract 2406: Identifying and targeting chemoresistant subclones in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressively metastatic subtype that can only be treated by chemotherapy. Nearly 50% of patients have residual disease after neoadjuvant chemotherapy (NACT) and have extremely poor prognoses. Recent genome sequencing studies have revealed extensive intratumoral heterogeneity (ITH) in treatment-naïve TNBC. However, the functional contribution of ITH to chemoresistance in TNBC is unknown. To understand how this occurs, we are generating patient derived xenograft (PDX) models from treatment-naïve TNBC in order to identify and characterize tumor cell populations that are responsible for chemoresistance with the ultimate goal of selectively targeting resistant tumor clones. Through an IRB-approved clinical trial using standard techniques, samples (primary tumor, skin metastasis, and blood for germline reference) were obtained from a patient with newly diagnosed, untreated metastatic TNBC. Subsequently, this patient was found to have disease resistant to chemotherapy. Tumor cells were implanted into the humanized mammary fat pad of NOD/SCID mice to establish PDX models of the primary (PIM1-P) and metastatic (PIM1-M) tumors. RNA sequencing and whole-exome sequencing (∼300X) were performed on the patient's primary and metastatic tumors and the first and third passage PDX tumors. Mouse sequences were computationally subtracted from the PDX data and which was then processed according to Genome Analysis Toolkit best practices workflow. Variants were called using MuTect and copy number alterations were estimated by ExomeCN. This revealed high concordance between the genomic profiles of the patient and PDX models. While there were 81 somatic non-silent mutations shared in the patient and PDX, only a few fell in known cancer genes, including TP53 (V143fs), ELF4 (L593H), and ARID3A (R351P). Modeling of clonal clusters with ABSOLUTE revealed ITH in the patient tumor that is preserved in the PDX. Both the patient and PDX model (PIM1-P) exhibited progressive disease when treated with paclitaxel. Only partial responses were observed in mice treated with doxorubicin plus cyclophosphamide (AC), consistent with residual disease after standard NACT for patients with TNBC. AC treatment resulted in an ∼60% reduction in tumor volume which was not enhanced by repeated cycles. Tumors rapidly re-grew when treatment was halted or if treated tumor cells were engrafted into the mammary fat pads of new recipient mice. This suggests that a subpopulation of AC-resistant tumor cells is present in PIM1-P tumors. To dissect this subpopulation, we established conditions for simultaneous tracking of thousands of PIM1-P tumor clones in vivo using a high-complexity library of up to 30 million unique DNA barcodes. Mice engrafted with barcoded PIM-1P tumors are being treated with AC to identify and characterize AC-resistant tumor cells present in PIM1. Citation Format: Gloria V. Echeverria, Sahil Seth, Shirong Cai, Stacy Moulder, William F. Symmans, Timothy P. Heffernan, Jeffrey Chang, Helen Piwnica-Worms. Identifying and targeting chemoresistant subclones in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2406.

Abstract 1631: FGF2 activation of FGFR1 in head and neck squamous cell carcinoma is associated with more invasive disease and can be attenuated by FGFR inhibition

Abstract Introduction. Head and neck squamous cell carcinomas (HNSCCs) account for nearly 600,000 deaths worldwide annually and have limited treatment options. Approximately 20% of HNSCCs harbor amplifications of fibroblast growth factor receptor 1 (FGFR1) on chromosome 8p, however FGFR1 amplification by itself does not predict clinical response to FGFR inhibitors. We hypothesized that FGF2, or basic FGF, ligand expression is a better marker of FGFR activation and predictor of response to FGFR inhibitors. Results. A tissue micro array (TMA) of HNSCC patient biopsies was stained and quantitated for FGF2 expression by Aperio ImageScope software. FGF2 was significantly increased in recurrent tissue samples (p = 0.04). We examined a number of immortalized HNSCC cell lines and found that overexpression of both FGF2 and FGFR1 predicted response to the selective FGFR inhibitor PD173074. FGFR inhibition did not cause apoptosis, but rather induced a G0/G1 arrest and growth inhibition. FGFR inhibition also induced a change in cell morphology, with a significant increase in cell size and adherence. The expression of epithelial-to-mesenchymal transition (EMT) proteins was examined and FGF2-FGFR1 activation was associated with a more mesenchymal phenotype. Accordingly, FGFR inhibition reversed invasiveness as measured using the Incucyte WoundMaker scratch assay, suggesting that HNSCCs with FGF2-FGFR1 activation have more metastatic potential. Invasiveness of these cells in vivo was confirmed using orthotopic injection into the buccal pad of NSG mice. Once primary tumors reached 0.8 cm in size, mice were sacrificed and buccal mucosa, lung, liver, and neck tissue were examined post-mortem. All of the injected animals developed local invasion, and distant metastases in the lungs. 5/7 mice also had metastases in the liver and this model is being used to test the ability of FGFR inhibition to prevent metastasis. The mechanism of autocrine FGF2-FGFR1 activation was further explored and FGF2 was found to be secreted in association with extracellular vesicles (ECVs). Interestingly, inhibition of FGFR reduced secretion of ECVs and FGF2, providing a novel approach to target autocrine and paracrine FGFR1 activation within the tumor. We further tested a number of small molecule inhibitors in combination with PD173074 to look for synergistic combinations of kinase inhibitors and found significant synergy between EGFR and FGFR inhibitors suggesting this combination may be most effective in patients with HNSCC. Conclusions. Increased FGF2 in HNSCC patient samples is correlated with recurrent disease. FGF2-FGFR1 activation increases invasiveness through activation of EMT genes both in vitro, and in an orthotopic model. Inhibition of FGF2-FGFR1 reversed the invasive phenotype in vitro and may be an effective therapeutic strategy to reduce metastases in HNSCC patients. Citation Format: Isabel A. English, Jacqueline Martinez, Edward El Rassi, Mark Schmidt, Ellen Langer, Sophia Bornstein, John Gleysteen, Melissa Wong, Brian Druker, Elie Traer. FGF2 activation of FGFR1 in head and neck squamous cell carcinoma is associated with more invasive disease and can be attenuated by FGFR inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1631.

Abstract 4275: Exploring the metastatic potential of exosomal NM23 signaling using a triple negative breast cancer model in mice

Abstract Decades of research has established the idea that cancer cells of the primary tumor prime the metastatic microenvironment prior to establishing metastases at distant sites in the body. While this process is thought to facilitate neocolonization of dislodged primary tumor cells, the mechanisms by which cancer cells communicate to their remote targets remain largely unknown. Our lab has previously shown that triple negative breast cancer cells release exosomes carrying NM23, a nucleoside diphosphate kinase (eNDPK) implicated in promoting angiogenesis and pro-metastatic events extracellularly. To further elucidate the role of eNDPK in breast cancer signaling, we are developing a novel murine model that will allow us to examine its effects in vivo, as well as establish a timeline for the occurrence of metastases. To establish our metastasis model in mice, we first showed that injecting human MDA-MB-231 cells into the mammary fat pad results in the formation of a primary tumor and subsequent development of metastases in the lung. Treating mice with inhibitors of NDPK activity reduces the size of the primary tumor and prevents secondary tumor formation in the lung. To mimic the pulmonary tumor microenvironment, we have isolated endothelial cells from the lungs of 6-8 day old mouse pups using magnetic beads conjugated to the endothelial cell markers CD54, CD102, or CD106. Purity of endothelial cells was confirmed by immunofluorescence staining. In preparation for in vivo experiments that demonstrate eNDPK targeting to lung endothelial cells, we have 3D printed biocompatible polylactic acid (PLA) tubular inserts that are capable of supporting lung endothelial cells encompassed in a gelatinous cell scaffold. Inserts containing lung endothelial cells are surgically introduced into the subcutaneous tissue of adult SCID mice. Human MDA-MB-231 cells expressing GFP-tagged tetraspanin are then injected orthotopically into the mammary fat pads of mice. After a period of 7-21 days, the inserts are excised and analyzed for the presence of GFP-labeled exosomes containing eNDPK and reorganization of the insert microenvironment. Using this model for metastasis, we will be able to confirm the involvement of eNDPK in cell-cell communication between primary tumor cells and their targets in vivo. The significance of uncovering the mechanism(s) by which cancer cells metastasize is emphasized by the fact that recurrence of cancer at distant sites is associated with the most negative outcome in women diagnosed with breast cancer. Implication of eNDPK signaling in metastasis will lead to future research in developing novel small molecule inhibitors. Further, eNDPK can be used as a biomarker for the beginning stages of metastatic breast cancer, replacing current unreliable early detection methods and improving long-term survival outcomes. Citation Format: Suzann Duan, Senny Nordmeier, Iain L.O. Buxton. Exploring the metastatic potential of exosomal NM23 signaling using a triple negative breast cancer model in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4275.

Abstract 4248: Functional imaging markers for blockade of breast cancer metastasis by IGF1R and insulin receptor targeted drugs using novel MRI and targeted iron oxide nanoparticles

Abstract IGF1R and insulin receptor (IR) regulate biology of estrogen positive (ER+) and triple negative breast cancer (TNBC) cells. The results of initial clinical trials with anti-IGF1R drugs have been disappointing due to feedback upregulation of insulin receptor (IR) signaling and absence of biomarkers for these drugs. We evaluated the effects of BMS-754807, a dual tyrosine kinase inhibitor of IGF1R/IR on ER+ and TNBC. In ER+ MCF-7 cells, it inhibited xenograft growth of MCF-7 tumors (n = 10 per treatment) compared to vehicle. Interestingly, while tumor growth was suppressed over a period of five weeks, eventually the tumors displayed resistance to BMS-754807. In TNBC, it inhibited motility in vitro. In contrast to ER+ cells, BMS-754807 did not inhibit primary tumor growth of TNBC cells injected into the mammary fat pad of mice. But at a dose of 50 mg/kg daily inhibited metastasis of TNBC cells, MDA-231-LM2 and MDA-MB435A/LCC6 cells, in the orthotopic and tail vein models of metastasis compared to vehicle (n = 10/group). Our data indicate that regulation of metastasis and tumor growth by IGF1R can be discrete events and functional imaging to identify biological properties of metastatic breast cancer regulated by IGF1R/IR are needed to better define treatments. While MRI is a powerful tool for detecting and imaging cancer, its utility in imaging metastasis to the lung is limited due to the challenges of lung MRI with conventional 3D gradient echo (GRE). MRI does not visualize lung well, mainly due to the abundance of air-tissue interfaces, which cause the MR signal to decay too rapidly for conventional MRI pulse sequences to capture. Clinically metastases are monitored by CT or PET but exposure of patients to ionizing radiation is a concern and problematic in longitudinal studies monitoring response to a targeted drug. Therefore, we recently reported the utility of a novel MR sequence called sweep imaging with Fourier transformation (SWIFT), where the data is acquired quasi-simultaneously with the radiofrequency pulse, to image lung metastasis of breast cancer. Here, we monitored response to IGF1R and IGF1R/IR targeted drugs in preclinical models of lung metastasis of breast cancer. We used MDA-231-LM2 cells with the tail vein injection model of metastasis. Mice with breast cancer metastases in the lungs were treated with either huEM164, an antibody against IGF1R, or BMS-754807. Metastasis was monitored by BLI and SWIFT MRI weekly. SWIFT was more sensitive in detecting inhibition of metastasis by these drugs. Thus, dual inhibition of IGF1R and IR is effective in blocking growth of ER+ and metastasis of TNBC. However, combination of this therapeutic strategy with other agents may be necessary to prevent or delay onset of resistance. Further, noninvasive biomarkers of response to IGF1R/IR targeted drugs can be developed with SWIFT imaging. Citation Format: Deepali Sachdev, Huy Donguyen, Naoharu Kobayashi, Sidath C. Kumarapperuma, Joeseph C. Weber. Functional imaging markers for blockade of breast cancer metastasis by IGF1R and insulin receptor targeted drugs using novel MRI and targeted iron oxide nanoparticles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4248.

Mammary Epithelial Cell Lineage Analysis via the Lyon's Hypothesis.

Implants of mammary glands from a single mammary fat pad in a H253 transgenic female mouse heterozygous for a lacZ-labeled X chromosome were analyzed at various time points following transplantation into the epithelium-cleared mammary fat pads of immune-compromised mice. The results show that the lacZ-marked X chromosome, demonstrated by nuclear-associated X-gal staining, was confined to a single epithelial clone that gave rise to the cap cells of all growing terminal end buds (TEB) in the expanding mammary outgrowths and also the basal cells of the elongated ducts. The nuclei of luminal cells in these ducts were uniformly negative for lacZ expression indicating that they were derived from cellular precursors that contained a silenced lac-Z marked X chromosome. This observation confirms the earlier work of Williams and Daniel, who concluded that cap cells were the precursors of the basal (myoepithelial cells) of the subtending mammary ducts.

Development of Wireless Power Transfer using Capacitive Method for Mouse Charging Application

Wireless power transfer (WPT) is a non-contact power transfer within a distance. With the advantage of not-contact concept, WPT enhances the flexibility movement of the devices. Basically, there are three types of the WPT which are inductive power transfer (IPT), Capacitive Power Transfer (CPT) and Acoustic Power Transfer (APT). Among these, capacitive power transfer (CPT) has the advantages of confining electric field between coupled plates, metal penetration ability and also the simplicity in circuit topologies. Therefore, we focus on the capacitive method in this paper. To be specific, this paper aims to develop a wireless mouse charging system using capacitive based method. This method enables wireless power transmission from mouse pad to a wireless mouse. Hence, no battery requires to power up the mouse. In this paper, a high efficiency Class-E converter is described in details to convert the DC source to AC and the compensation circuit of resonant tank is also proposed at the transmitter side in order to improve the efficiency. In the end, a prototype is developed to prove the developed method. The performances analysis of the developed prototype is discussed and the future recommendation of this technique is also presented.

Enhanced anti-tumor activity and cytotoxic effect on cancer stem cell population of metformin-butyrate compared with metformin HCl in breast cancer.

Metformin, which is a drug commonly used to treat type 2 diabetes, has shown anti-tumor effects in numerous experimental, epidemiologic, observational, and clinical studies. Here, we report a new metformin derivative, metformin-butyrate (MFB). Compared to metformin-HCl, it more potently activates AMPK, inhibits mTOR, and impairs cell cycle progression at S and G2/M phases. Moreover, MFB inhibits the mammosphere formation of breast cancer cells and shows cytotoxic effects against CD44+CD24−/low populations in vitro and in vivo, indicating that it might have preferential effects on the cancer stem cell population. MFB showed synergistic cytotoxicity with docetaxel and cisplatin, and MFB pretreatment of breast cancer cells prior to their injection into the mammary fat pads of mice significantly decreased the obtained xenograft tumor volumes, compared with untreated or metformin-pretreated cells. Overall, MFB showed greater anti-neoplastic activity and greater efficacies in targeting the G2/M phase and breast cancer stem cell population, compared to metformin-HCl. This suggests that MFB may be a promising therapeutic agent against aggressive and resistant breast cancers.

VEGF-D-enhanced lymph node metastasis of ovarian cancer is reversed by vesicular stomatitis virus matrix protein.

Lymphatic metastasis is a poor prognostic factor in ovarian cancer, which correlates to the majority of cancer deaths. Matrix protein (MP) of vesicular stomatitis virus (VSV) exhibits potent antitumor and antiangiogenic activities through inducing apoptosis and inhibiting angiogenesis. In this study, the antitumor and antimetastatic effects of MP were further investigated. Wild-type SKOV3 (WT-SK) cells were successfully transfected with empty vector pcDNA3.1 plasmid, or pcDNA3.1-VEGF-D recombinant plasmid to construct cell lines named EV-SK, and VEGFD-SK, respectively. Inhibition of VEGFD-SK cell migration and invasion was detected by Transwell and wound healing assay. Then, lymphogenous metastatic model of ovarian cancer was established by injecting VEGFD-SK cells subcutaneously into the left hindlimb claw pad of nude mice. The inducted apoptotic effect of MP on VEGFD-SK cells were assessed by flow analysis and Hoechst-33258 staining, respectively, in vitro. The in vivo antitumor and antiangiogenic activities of MP gene were evaluated with lymphogenous metastatic model of ovarian cancer. Tumor volume and lymphatic metastasis rates were measured. Lymphatic vessels were delineated using Evan's blue and LYVE-1 staining. Expression of VEGF-D and MMP-2 were evaluated by immunostaining. Apoptosis of tumor cells was analyzed by Hoechst-33258 staining. Mice bearing VEGFD-SK tumor cells displayed more rapid tumorigenesis, higher lymphogenous metastatic tendency and increased lymphatic vessel density compared with the mice bearing WT-SK or EV-SK cells. However, VEGF-D-enhanced metastasis was evidently reversed by MP. MP significantly reduced the invasion of VEGFD-SK cells, tumor volume, lymphatic metastasis rates and lymphatic vessel density compared with control groups (P<0.05), accompanied with down-expression of VEGF-D and MMP-2 and increased apoptosis. Our data indicate that MP has strong antitumor and antimetastatic abilities, and it may be a promising therapeutic strategy against the lymphatic metastasis of human ovarian cancer.

CD24 cell surface expression in Mvt1 mammary cancer cells serves as a biomarker for sensitivity to anti-IGF1R therapy

BackgroundThe pro-tumorigenic effects of the insulin-like growth factor receptor (IGF1R) are well described. IGF1R promotes cancer cell survival and proliferation and prevents apoptosis, and, additionally it was shown that IGF1R levels are significantly elevated in most common human malignancies including breast cancer. However, results from phase 3 clinical trials in unselected patients demonstrated lack of efficacy for anti-IGF1R therapy. These findings suggest that predictive biomarkers are greatly warranted in order to identify patients that will benefit from anti-IGF1R therapeutic strategies.MethodsUsing the delivery of shRNA vectors into the Mvt1 cell line, we tested the role of the IGF1R in the development of mammary tumors. Based on CD24 cell surface expression, control and IGF1R-knockdown (IGF1R-KD) cells were FACS sorted into CD24− and CD24+ subsets and further characterized in vitro. The tumorigenic capacity of each was determined following orthotopic inoculation into the mammary fat pad of female mice. Tumor cells were FACS characterized upon sacrifice to determine IGF1R effect on the plasticity of this cell’s phenotype. Metastatic capacity of the cells was assessed using the tail vein assay.ResultsIn this study we demonstrate that downregulation of the IGF1R specifically in cancer cells expressing CD24 on the cell surface membrane affect both their morphology (from mesenchymal-like into epithelial-like morphology) and phenotype in vitro. Moreover, we demonstrate that IGF1R-KD abolished both CD24+ cells capacity to form mammary tumors and lung metastatic lesions. We found in both cells and tumors a marked upregulation in CTFG and a significant reduction of SLP1 expression in the CD24+/IGF1R-KD; tumor-suppressor and tumor-promoting genes respectively.Moreover, we demonstrate here that the IGF1R is essential for the maintenance of stem/progenitor-like cancer cells and we further demonstrate that IGF1R-KD induces in vivo differentiation of the CD24+ cells toward the CD24- phenotype. This further supports the antitumorigenic effects of IGF1R-KD, as we recently published that these differentiated cells demonstrate significantly lower tumorigenic capacity compared with their CD24+ counterparts.ConclusionsTaken together these findings suggest that CD24 cell surface expression may serve as a valuable biomarker in order to identify mammary tumors that will positively respond to targeted IGF1R therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0711-7) contains supplementary material, which is available to authorized users.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

In Vivo Visualization and Characterization of Epithelial-Mesenchymal Transition in Breast Tumors.

The activation of the epithelial-to-mesenchymal transition (EMT) program is a critical step in cancer progression and metastasis, but visualization of this process at the single-cell level, especially in vivo, remains challenging. We established an in vivo approach to track the fate of tumor cells based on a novel EMT-driven fluorescent color switching breast cancer mouse model and intravital two-photon laser scanning microscopy. Specifically, the MMTV-PyMT, Rosa26-RFP-GFP, and Fsp1-Cre triple transgenic mouse model was used to monitor the conversion of RFP-positive epithelial cells to GFP-positive mesenchymal cells in mammary tumors under the control of the Fsp1 (ATL1) promoter, a gate-keeper of EMT initiation. RFP-positive cells were isolated from the tumors, sorted, and transplanted into mammary fat pads of SCID mice to monitor EMT during breast tumor formation. We found that the conversion from RFP- to GFP-positive and spindle-shaped cells was a gradual process, and that GFP-positive cells preferentially localized close to blood vessels, independent of tumor size. Furthermore, cells undergoing EMT expressed high levels of the HGF receptor, c-Met, and treatment of RFP-positive cells with the c-Met inhibitor, cabozantinib, suppressed the RFP-to-GFP conversion in vitro Moreover, administration of cabozantinib to mice with palpable RFP-positive tumors resulted in a silent EMT phenotype whereby GFP-positive cells exhibited reduced motility, leading to suppressed tumor growth. In conclusion, our imaging technique provides a novel opportunity for visualizing tumor EMT at the single-cell level and may help to reveal the intricacies underlying tumor dynamics and treatment responses. Cancer Res; 76(8); 2094-104. ©2016 AACR.

The Fate of Fat Grafts in Different Recipient Areas: Subcutaneous Plane, Fat Pad, and Muscle.

Although fat is transplanted into several layers, including subcutaneous, fat, and muscle layers, there is little (clinical) scientific basis for these procedures. To determine the optimal recipient layer for fat transplantation. Fat harvested from inguinal fat pads of green fluorescent protein (GFP) mice was grafted into the subcutaneous and intramuscular planes and the fat pads of C57 mice. Specimens collected after 1, 4, 8, 12, and 16 weeks were stained with hematoxylin and eosin to determine angiogenesis and fibrosis in the grafts. The survival rate of donor adipose tissue was determined by measuring GFP expression. Fat was retained longer in fat pads than in subcutaneous layers of recipient mice and longer in subcutaneous than in intramuscular layers. Angiogenesis and vascularized connective tissue were greater in intramuscular than in subcutaneous or fat grafts. Neovascularization, however, was similar in fat pads and subcutaneous grafts. Survival rate was higher for intramuscularly injected fat than subcutaneously and fat pad injected fat. Fat pad injection showed the highest graft retention rate, indicating that fat pads maybe the optimal area for fat transplantation. The increased blood supply to muscle suggests that intramuscular injection maybe optimal when there is little movement.

Gesture and Voice Based PC Control Using Real Time Camera

Human Computer Interaction with hand gesture has become increasingly popular today. The physical mouse device whether it is our basic desktop mouse or laptop touch pad, it require physical contact of user to convey input. Many advanced technologies have came but they all require physical contact. Hence we need a ubiquitous interface to do this. Our method is to use a camera device and computer gesture and voice based technology, like image segmentation as well as gesture recognition, to control mouse actions such as left click, double click, right click and dragging. We can show how it can do the things that present mouse devices can do. Our project Gesture and Voice Based PC Control is a real time system capable of understanding mouse commands given by hand gestures and voice commands. The end user is able to communicate with computer with the commands given by hand gestures and voice commands. This will avoid the need of physical contact to the computer to control mouse inputs. In this way the interface becomes ubiquitous. Introduction As computer technology continues to improve, people are having smaller and smaller electronic. Devices. They want to use these devices ubiquitously. There is need of a new interfaces designed specifically for use with these electronics devices. We are increasingly recognizing the importance of HCI, and in particularly gesture based gesture recognition. Simple interfaces already exist, such as embedded keyboard, folder keyboard and mini keyboard [2] . However, these interfaces need some amount of space to use as well as can’t be used in motion. Touch screens devices are also providing a good control interface and nowadays it is used globally in many applications [3] . But the cost and other hardware limitations makes it limited. With application of gesture technology and controlling the mouse actions by natural hand gestures, we can reduce the space required. We propose a good approach that uses a camera to control the mouse actions. Problem Definition To develop a software solution to a problem, the first step is to understand the problem. Mouse is a physical device, subject to mechanical wear and tear also demands the user to make physical contact to convey his Input, which is no longer appropriate. Natural actions in human to human communication, such as speak and gesture, seem more appropriate. Interfaces based on computational perception and computer gesture should be available for accomplishing the goals of ubiquitous computing [5] . The problem here is to develop a way so that humans can interact with a computer without having any physical contact with the computer. Need for the New System Mouse is a physical device. This device is subject to mechanical wear and tear. It requires the user to make physical contact to convey his Input, which is not appropriate. Instead, natural actions in human-to-human communication, such as speak and gesture, seem more appropriate [5] . This