Metastatic Breast Cancer Model Research Articles

Abstract 4264: Mathematical modeling of differential effects of sunitinib on primary tumor and metastatic growth

Abstract Sunitinib is a drug with anti-angiogenic activity used in the treatment of patients with metastases from renal cell carcinoma or gastrointestinal tumors. However, despite clear efficacy in reducing established tumor growth, recent preclinical studies have shown limited, or even opposing, efficacies in preventing metastatic spread. In this work, we evaluated a mathematical metastatic model to describe primary tumor and metastatic dynamics in response to sunitinib in a clinically relevant mouse model of spontaneous metastatic breast cancer that develops after surgical removal of an orthotopically implanted primary tumor. Mice received either vehicle or sunitinib in the neoadjuvant (presurgical) setting according to different schedules. The experimental dataset comprises measurements of primary tumor kinetics, metastatic burden and pre-surgical molecular and cellular biomarkers, including vascular cell Ki67 and CD31 expression, circulating tumor cells (CTCs) and myeloid derived suppressor cells (MDSCs). Estimation of the model's parameters was performed using a mixed-effects population approach. To describe tumor growth under sunitinib treatment, a simple ordinary differential equation model of tumor growth inhibition was used. Population fits obtained modeling the effect of treatment only on primary tumor growth described well the experimental data of all the treated groups considered. On the contrary, simulations of treatment also on metastasis could not reproduce the behavior of the data. By inserting in the model the available biomarkers as covariates, through a Wald-test, measurements of Ki67+/CD31+, CTCs and granulocytic MDSCs were found significantly correlated with the model parameter expressing the metastatic aggressiveness of the tumor. Together, these mathematical models confirm a differential effect of sunitinib on primary (localized) tumors compared to secondary (metastatic) disease. Our results suggest that Ki67+/CD31+, CTCs and MDSCs measurements might help in predicting metastatic potential and thus aid in predicting benefit in overall survival for preoperative antiangiogenic treatments. Citation Format: Chiara Nicolò, Michalis Mastri, Amanda Tracz, John M. Ebos, Sébastien Benzekry. Mathematical modeling of differential effects of sunitinib on primary tumor and metastatic growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4264.

Abstract 950: New strategy to sensitize pancreatic and bladder cancer to gemcitabine

Abstract Pancreatic Ductal Adenocarcinoma (PDAC), the major histological subtype of pancreatic cancer displays few symptoms for early detection and the majority of PDAC patients are diagnosed with advanced or metastatic disease. PDAC patients with advanced disease lack durable responses to chemotherapy and have a dismal prognosis. Gemcitabine is the cornerstone of care for advanced and metastatic PDAC. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. Accordingly, strategies that lead to upregulation of signaling pathways and enzymes involved in generating the active metabolite may lead to improved outcome in PDAC. We have developed a potent, selective, small molecule inhibitor of casein kinase-1 delta (CK1δ) and demonstrated that inhibition of CK1δ activity specifically compromises the growth, survival and invasion of breast cancer cells that overexpress CK1δ. Notably, our lead compound, SR-3029 provokes pre-clinical tumor regression of triple negative breast cancer (TNBC), including lung metastatic TNBC and basal-like PDX breast cancer models. We observed that CK1δ is upregulated in other cancers including bladder cancer and demonstrated that SR-3029 acts in synergy with DNA damaging cytotoxic agents. Notably, the combination of gemcitabine with SR-3029 resulted in synergistic antiproliferative activity and impaired colony formation in both pancreatic and bladder cancer cells. Mechanistic studies demonstrate that silencing CK1δ or inhibition of CK1δ activity by SR-3029 induces a significant upregulation of deoxycytidine kinase (DCK; rate limiting enzyme for gemcitabine activation) both at the RNA and protein levels, which results in increased cellular DCK and its active gemcitabine metabolite. We evaluated the effect of SR-3029 alone and in combination with gemcitabine in an orthotopic pancreatic tumor mouse model and observed much improved tumor growth inhibition with combination treatment, concomitant with increased expression of DCK. In conclusion, our study suggests that CK1δ plays signaling roles in DNA metabolism and the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic pancreatic cancer as well as other cancers. Citation Format: Simon Bayle, Francesca Vena, Andrii Monastyrskyi, William Roush, Derek Duckett. New strategy to sensitize pancreatic and bladder cancer to gemcitabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 950.

Abstract 3726: Design of personalised polymer based combination therapeutics for advanced stage breast cancer patients

Abstract Our objective is to engineer tumour-targeted polymer-based combination therapies specifically designed to treat metastatic breast cancer (BC) in a personalised manner. Our strategy is to develop novel multicomponent polymer conjugates and assess structure activity relationships in clinically relevant models to understand mechanisms of action. Furthermore, we are searching for novel drug combinations, including tumour-derived exosome release pathway inhibitors due to their association with metastasis and tumour drug resistance mechanisms. NCA polymerization techniques have allowed us to precisely control the synthesis of well-defined star-based (STP) and linear (LTP) polypeptidic architectures. After fluorescence labelling, we studied our systems in vitro demonstrating a much more rapid uptake for STP through clathrin-coated mediated endocytosis. pK and biodistribution in healthy mice revealed renal excretion profiles and greater terminal and accumulation half-lives for STP architectures when compared to the LTP. Remarkably, we also observed clear accumulation in immune system-related organs including the spleen and lymph nodes (LN) (up to 40% ID/g tissue accumulation in the LN after i.v. administration). This is possibly due to their inherent structural and morphological features, such as their size (≈100nm), highly negative z-potential values, and/or hydrophilic surfaces. These data highlight the great potential of our stabilized self-assemblies as carriers to target LN metastasis, cancer immunotherapy, or immune system-related approaches, such as vaccination. In parallel, we have performed a High Throughput Screening to select synergistic drug combinations to be used in polymer-based combination approaches through rationally designed linkers that confer adequate drug release kinetics. To perform this approach we selected four metastatic human BC cell lines representing clinical BC subtypes. All cell models have been fully characterized regarding their Cathepsin B activity, intracellular pH, as well as oestrogen, progesterone, Her2 receptors, GSH and exosomes levels; all representing patient-specific biomarkers. Cell viability and exosomes release modulation have been studied following treatments and several drug combinations have been selected for each specific BC subtype. With selected drug combinations different linking chemistry has been designed. We have studied a combination conjugate with the chemotherapeutic agent conjugated to PGA through two different length pH-labile hydrazone linkers. This provided different therapeutic outputs in cells and in a metastatic immunocompetent orthotopic breast cancer model, not only for the primary tumor but also for metastasis progression. The results obtained so far open up a wide range of opportunities for the currently unsuccessful clinical approaches to target LN metastasis and cancer immunotherapy. Citation Format: Ana Armiñán, Zoraida Andreu, Juan J Arroyo-Crespo, David Charbonnier, Esther Masiá, Fernanda Rodriguez-Otomin, Aroa Duro-Castano, Vicent J Nebot, Maria J Vicent. Design of personalised polymer based combination therapeutics for advanced stage breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3726.

Alterations in NO- and PGI2- dependent function in aorta in the orthotopic murine model of metastatic 4T1 breast cancer: relationship with pulmonary endothelial dysfunction and systemic inflammation

BackgroundPatients with cancer develop endothelial dysfunction and subsequently display a higher risk of cardiovascular events. The aim of the present work was to examine changes in nitric oxide (NO)- and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the formation of lung metastases and to local and systemic inflammation in a murine orthotopic model of metastatic breast cancer.MethodsBALB/c female mice were orthotopically inoculated with 4T1 breast cancer cells. Development of lung metastases, lung inflammation, changes in blood count, systemic inflammatory response (e.g. SAA, SAP and IL-6), as well as changes in NO- and PGI2-dependent endothelial function in the aorta, were examined 2, 4, 5 and 6 weeks following cancer cell transplantation.ResultsAs early as 2 weeks following transplantation of breast cancer cells, in the early metastatic stage, lungs displayed histopathological signs of inflammation, NO production was impaired and nitrosylhemoglobin concentration in plasma was decreased. After 4 to 6 weeks, along with metastatic development, progressive leukocytosis and systemic inflammation (as seen through increased SAA, SAP, haptoglobin and IL-6 plasma concentrations) were observed. Six weeks following cancer cell inoculation, but not earlier, endothelial dysfunction in aorta was detected; this involved a decrease in basal NO production and a decrease in NO-dependent vasodilatation, that was associated with a compensatory increase in cyclooxygenase-2 (COX-2)- derived PGI2 production.ConclusionsIn 4 T1 metastatic breast cancer in mice early pulmonary metastasis was correlated with lung inflammation, with an early decrease in pulmonary as well as systemic NO availability. Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway.

Validation of Cdc42 Inhibition as the Mechanism of Action of the Anti‐metastatic Drug MBQ‐167, Using Budding Yeast as a Model System

Cdc42 (Cell division control protein 42 homolog), a Rho GTPase, is highly conserved among eukaryotes in its structure and function. Mechanical or chemical cues in the microenvironment stimulate the localized activation of Cdc42 to rearrange the actin cytoskeleton and regulate cell polarity and motility. Its role in cell polarization was first discovered in the budding yeast Saccharomyces cerevisiae, but was subsequently shown to also establish directional motility in animal cells. Accordingly, in cancer, Cdc42 promotes migration, invasion, and spread of tumor cells. Thus, we targeted Cdc42 as a therapeutic strategy to treat metastatic breast cancer, and designed and characterized the small molecule MBQ‐167 as a potent inhibitor against Cdc42 and the homolog Rac. We recently reported that MBQ‐167 inhibits cancer cell and tumor growth and spread in‐vitro and in‐vivo using a mouse xenograft model of metastatic breast cancer. Since budding yeast express a single Cdc42 gene and do not express Rac, we used this well characterized model of polarization to validate the MBQ‐167 specificity for Cdc42 inhibition. Using a tetracycline regulated transcriptional inactivation (Tet‐Off) system in the promoter region of the yeast CDC42 gene, we knocked‐down Cdc42 supplementing the media with doxycycline (DOX; IC=10μg/μL). Next, we analyzed the growth, budding pattern, and Cdc42 activity of yeast exposed to DOX, MBQ‐167, or in combination. As expected, maximum growth, doubling time and budding polarity of yeast with the Tet‐Off system were reduced when treated with DOX (EC50=10μg/mL). The same effect was seen with MBQ‐167 (ED50=100μM) alone and in combination with DOX. Ultimately, pull‐downs of the active Cdc42‐GTP complex confirmed our hypothesis that MBQ‐167 disrupts cell polarity through impeding Cdc42 activation. Hence, we have validated MBQ‐167 as a Cdc42 inhibitor in another biological context and present a method to screen Cdc42 inhibitors as potential therapeutics against cancer metastasis.Support or Funding InformationThis work is partially supported by grants from the Susan G Komen for the Cure and the Puerto Rico Science and Technology Research Trust (S.D.), NIH/NIGMS P20GM103475 (JRRM), and NIH/NIGMS‐RISE R25 GM061838 (MJR)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel Rac Inhibitors as Targeted Therapeutics for Metastatic Breast Cancer

Metastatic disease is the primary cause of breast cancer mortality, due to the lack of effective therapy. The Rho GTPase Rac is integral for the promotion of cancer cell migration/invasion, proliferation, and survival. Since metastatic breast cancers often overexpress or exhibit high Rac activity, inhibition of Rac is a viable strategy against metastatic cancer. Recently, we characterized EHop‐016, a small molecule that inhibits Rac activity of metastatic breast cancer cells with an IC50 of 1 μM. EHop‐016, which is 10–100 times more active than previously available Rac inhibitors, inhibits the activity of the Rac downstream effector p21 activated kinase (PAK), lamellipodia extension, and cell migration of metastatic breast cancer cells. Moreover, EHop‐016 at ≥ 25 mg/kg Body Weight (BW) significantly reduced tumor growth, metastasis, and angiogenesis in a mouse model. However, our recent pharmacokinetic study of EHop‐016 in a mouse model demonstrated that its bioavailability should to be improved for further pharmacological development. Our hypothesis is that improvement of the EHop‐016 structure will provide probes with increased potency against Rac and improved bioavailability. Herein we have tested several Ehop‐016 derivatives for their effects on breast cancer cell viability, cell migration, and Rac activation. Using MTT assays we found that the Ehop‐016 derivatives, HV‐107 and HV‐118, significantly inhibit the viability of metastatic breast cancer cell lines MDAMB‐231 and MDA‐MB‐435; while presenting minimal toxicity towards non‐cancerous mammary epithelial cells. The effects of HV‐107 and HV‐118 on the inhibition of Rac activation were tested by ELISA‐based Rac activity assays and pulldown assays. Results show that at 250 nM, HV‐107 inhibits Rac activation by 55%, whereas HV‐118 has a similar effect at 100 nM in MDA‐MB‐231 and MDA‐MB‐435 cells. Moreover, using transwell assays we studied the effects of both Rac inhibitors in metastatic breast cancer cell migration and observed a significant reduction. Finally, we tested HV‐107 in a mouse model of metastatic breast cancer and found a 25% reduction in tumor growth in mice treated with 5 mg/kg BW HV‐107. Taken together, our findings suggest HV‐107 and HV‐118 as promising Ehop‐016 derivatives with potential as anti‐metastatic agents, that should be further characterized.Support or Funding InformationThis study was supported by awards from the Susan Komen for the Cure, NIH/NIMHHD U54MD008149, and the Puerto Rico Science and Technology Trust to SD; NIH/NCRR R25GM061838 to UPR MSC; NIH/NIMHHD RCMI 8G12MD007583RCMI, Title V PPOHA 031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education to UCC; and PRINBRE (NIH/NIGMS P20GM103475‐13) Sub‐Award to LCP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiscale optical imaging of rare-earth-doped nanocomposites in a small animal model.

Rare-earth-doped nanocomposites have appealing optical properties for use as biomedical contrast agents, but few systems exist for imaging these materials. We describe the design and characterization of (i) a preclinical system for whole animal in vivo imaging and (ii) an integrated optical coherence tomography/confocal microscopy system for high-resolution imaging of ex vivo tissues. We demonstrate these systems by administering erbium-doped nanocomposites to a murine model of metastatic breast cancer. Short-wave infrared emissions were detected in vivo and in whole organ imaging ex vivo. Visible upconversion emissions and tissue autofluorescence were imaged in biopsy specimens, alongside optical coherence tomography imaging of tissue microstructure. We anticipate that this work will provide guidance for researchers seeking to image these nanomaterials across a wide range of biological models.

Characterizing the efficacy of cancer therapeutics in patient-derived xenograft models of metastatic breast cancer.

Basal-like breast cancers are aggressive and often metastasize to vital organs. Treatment is largely limited to chemotherapy. This study aims to characterize the efficacy of cancer therapeutics in vitro and in vivo within the primary tumor and metastatic setting, using patient-derived xenograft (PDX) models. We employed two basal-like, triple-negative PDX models, WHIM2 and WHIM30. PDX cells, obtained from mammary tumors grown in mice, were treated with twelve cancer therapeutics to evaluate their cytotoxicity in vitro. Four of the effective drugs-carboplatin, cyclophosphamide, bortezomib, and dacarbazine-were tested in vivo for their efficacy in treating mammary tumors, and metastases generated by intracardiac injection of tumor cells. RNA sequencing showed that global gene expression of PDX cells grown in the mammary gland was similar to those tested in culture. In vitro, carboplatin was cytotoxic to WHIM30 but not WHIM2, whereas bortezomib, dacarbazine, and cyclophosphamide were cytotoxic to both lines. Yet, these drugs were ineffective in treating both primary and metastatic WHIM2 tumors in vivo. Carboplatin and cyclophosphamide were effective in treating WHIM30 mammary tumors and reducing metastatic burden in the brain, liver, and lungs. WHIM2 and WHIM30 metastases showed distinct patterns of cytokeratin and vimentin expression, regardless of treatment, suggesting that different tumor cell subpopulations may preferentially seed in different organs. This study highlights the utility of PDX models for studying the efficacy of therapeutics in reducing metastatic burden in specific organs. The differential treatment responses between two PDX models of the same intrinsic subtype, in both the primary and metastatic setting, recapitulates the challenges faced in treating cancer patients and highlights the need for combination therapies and predictive biomarkers.

Immunotherapy approach with recombinant survivin adjuvanted with alum and MIP suppresses tumor growth in murine model of breast cancer

ABSTRACTSurvivin has received attention as a potential target for cancer immunotherapy because of its crucial role in oncogenesis. We undertook this study to evaluate the immunotherapeutic potential of combination of recombinant survivin along with adjuvant alum and immune modulator Mycobacterium indicus pranii (MIP). In vivo efficacy of the combination was studied in an invasive murine breast cancer model. Recombinant survivin protein was purified from Escherichia coli based expression system and characterized by western blotting. Purified survivin protein was combined with alum and MIP and was used for immunization of Balb/c mice. Antigen-primed animals were then challenged with syngeneic mammary tumor cells known as 4T-1. Balb/c mice spontaneously develop tumor when inoculated with 4T-1 cells. Antigen and adjuvant combination was immunogenic and significantly suppressed tumor growth in mice immunized with combination of recombinant survivin (10 µg), alum, and MIP. This is the first report that describes a combination immunotherapy approach using recombinant survivin, alum, and MIP in highly metastatic murine breast cancer model and holds promise for development of new biotherapeutics for cancer.

Abstract P5-05-06: Development of advanced pre-clinical in vivo models of metastatic breast cancer

Abstract Backgrounds: The fact that we continue to lose 40,000 women with breast cancer every year in the US despite the recent advance in basic research clearly demonstrate disconnect in translation of basic research findings to clinic. This is largely due to lack of appropriate animal model that mimic clinical conditions for preclinical studies that result in high failure rate of clinical trials. To date, we had established many syngeneic mouse models, which are not free from limitations; 1) few clinically relevant animal models with bone metastasis have been established, 2) syngeneic mouse model cannot address human cancer genomics and tumor heterogeneity. Patient-Derived Xenograft (PDX) model has emerged as pre-clinical model to address these issues, however, it suffers low tumor take rate of around 20-40%, and lack metastatic model. Here, we describe development of orthotopic implantation, and bone and liver metastatic breast cancer mouse models to overcome these limitations. Methods: 1) 4T1.2-luc3 cells that has metastatic potential to the bone were orthotropically inoculated as a syngeneic mouse model, imaged with IVIS and MRI. 2) Patient tumor tissues of 1mm(3) were implanted surgically into dorsal subcutaneous space (SQ), or orthotropically into mammary fat pat #2 and #4 (MFP). Results: 1) We established a syngeneic breast cancer bone metastasis model. Primary tumors were surgically resected days after 4T1.2-luc3 cells were orthotopically implanted under direct vision. Removal of primary tumor allowed bioluminescent visualization and quantification of bone metastasis by IVIS. We found that MRI was effective in evaluating bone metastasis and bone related events in these mice. MRI allows differentiation of bone metastasis from metastasis to the surrounding organs with bone destruction image, whereas conventional bioluminescence imaging shows only existence of cancer cells. 2) The overall tumor take rate of the tumor in PDX model was 46.0% (74/161 implantation site). Take rate from triple-negative breast cancer tumors was 56.1% (74/132), on the other hand, that from ER positive tumors was 0% (0/39). Tumor take rate was significantly better in MFP implantation than SQ (39.5%, 30/76 vs 51.2%, 44/85, p<0.01). Tumor weight were significantly heavier in MFP compared to SQ (0.072g vs 0.328g, p<0.00001). With more passage, the difference in tumor weight between SQ and MFP was significantly increased(p<0.0001). Finally, we developed a PDX breast cancer liver metastasis model by surgically implanting tissue fragment into liver using direct vision technique. We found MRI to be very useful as a living imaging modality to evaluate cancer progression in the deeply located metastatic sites of PDX models. Conclusions: We have established orthotropic syngeneic breast cancer bone metastasis model as well as improved breast cancer PDX model with synchronous liver metastasis utilizing MRI. Our novel models could be powerful tools for preclinical studies. Citation Format: Okano M, Kawaguchi T, Okano I, Katsuta E, Takabe K. Development of advanced pre-clinical in vivo models of metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-06.

Abstract P1-01-20: Adjuvant statin therapy efficacy is dictated by tumor dormancy and statin lipophilicity in ex vivo and in vivo models of metastatic breast cancer

Abstract Metastasis in breast cancer patients heralds mortality, as disseminated disease is generally chemoresistant. After tumor cells reach the ectopic tissue, they undergo an epithelial reversion to enter a period of quiescence, termed dormancy, which may last for decades before outgrowing again as mesenchymal/dedifferentiated masses. Thus, long-term, relatively non-toxic interventions that prevent metastatic outgrowth are needed to treat this mortal stage of tumor progression. Epidemiological analyses have suggested that statin usage, for cardiovascular indications, is correlated with a reduction in clinically-evident metastatic (though not in incidence of primary) breast cancer. The goal of this study is to demonstrate this is due to statins suppressing breast cancer cell proliferation and keeping the micrometastases in the dormant state. We have found that atorvastatin and simvastatin limit the growth of some cancer cell lines, but not others. The sensitive lines were marked by lacking surface E-cadherin, the hallmark of the mesenchymal phenotype. When E-cadherin is downregulated on epithelial tumor cells, the cells become growth inhibited by the statins. Furthermore, this is a direct effect, as we now have shown that hydrophilic statins are relatively ineffective compared to the membrane permeant lipophilic statins as tumor cells generally lack the transporters that enable these drugs to gain access to the cells. To determine whether the statins target the emergent metastatic tumor cells, we are using an all human microphysiological system (MPS) of the most common site for metastases, the liver. Briefly, a micro-hepatic tissue is established by seeding primary human liver cells in a porous scaffold subject to a physiological flow. RFP-labeled breast cancer cells are seeded into these microtissues and examined weeks later. Liver function and health are monitored by clinical chemistry assays performed on supernatant samples. We have previously shown that this system robustly reproduces tumor dormancy. Initial studies suggest that statins suppress the emergence of dormant tumor cells when challenged by stressors that lead to outgrowth. Additionally, atorvastatin suppresses proliferation of mesenchymal but not epithelial breast cancer cells in intrasplenic and mammary fat pad injection models for breast cancer metastasis to the liver and lung respectively. As 26% of adults currently take a statin for other medical conditions, these studies may suggest the best statin to use in the context of maintaining breast cancer dormancy long-term and delaying or avoiding the morbid emergence. Citation Format: Beckwitt CH, Clark AM, Warita K, Oltvai ZN, Wells A. Adjuvant statin therapy efficacy is dictated by tumor dormancy and statin lipophilicity in ex vivo and in vivo models of metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-20.

Methanolic Extract of Red Betel Leaves (Piper crocatum Ruiz & Pav) Perform Cytotoxic Effect and Antimigration Activity toward Metastatic Breast Cancer

Breast cancer is one type of cancer with a high mortality rate due to its metastatic property. Red betel leaves (Piper crocatum Ruiz dan Pav) has been known as herbal medicine containing biophenolic, such as apigenin and luteolin derivatives which has cytotoxic activity toward cancer cells. This study is intended to explore the inhibitory effect of Piper crocatum leaves methanolic extract (PCM) on cell proliferation and migration by using 4T1 cells as model of metastatic breast cancer. By using MTT assay, PCM performed cytotoxic activity in a dose dependent manner with IC50 value of 120 μg/mL. Wound healing assay revealed that migration inhibitory activity of PCM on 4T1 cells at the concentration of 30 µg/mL. In conclusion, PCM perform cytotoxic effect and antimigration activity toward metastatic breast cancer cells.Keywords : breast cancer cells, Piper crocatum Ruiz & Pav, cytotoxic, cell mgration

5-Chloro-2,4-dihydroxypyridine, CDHP, prevents lung metastasis of basal-like breast cancer cells by reducing nascent adhesion formation.

A drug for metastasis prevention is necessary. The orally administered anticancer drug S‐1 contributes to cancer therapy. In a mouse xenograft model of metastatic breast cancer from our previous study, the administration of S‐1 inhibited lung metastasis. However, the mechanism of inhibition remains elusive. S‐1 contains 5‐chloro‐2,4‐dihydroxypyridine (CDHP), which does not have the antigrowth activity, but prevents the degradation of 5‐fluorouracil, an anticancer reagent. In this study, we found that CDHP treatment shrinks cell morphology in metastatic basal‐like breast cancer cell lines. Wound healing assays showed reduced cell migration in CDHP‐treated cells. At the molecular level, CDHP treatment reduced the number of nascent adhesions, whereas the number of mature focal adhesions was not changed. These findings indicate that CDHP impairs focal adhesion formation, which results in a reduction in cell migration. For the in vivo metastasis assay, we used a highly lung‐metastatic cell line. We xenografted them into immunodeficient mice, and administered CDHP. To determine whether CDHP prevents metastasis, we measured the weights of harvested lungs. The results showed that the lung weights of the CDHP‐treated animals were not significantly different compared to the no‐tumor controls, whereas the vehicle group showed a number of metastatic foci and an increase in lung weight. These observations indicate that CDHP administration prevents metastasis. This study reveals a novel effect of CDHP for lung metastasis prevention. Our findings may facilitate the establishment of future metastasis prevention therapies.

Radionuclides transform chemotherapeutics into phototherapeutics for precise treatment of disseminated cancer

Most cancer patients succumb to disseminated disease because conventional systemic therapies lack spatiotemporal control of their toxic effects in vivo, particularly in a complicated milieu such as bone marrow where progenitor stem cells reside. Here, we demonstrate the treatment of disseminated cancer by photoactivatable drugs using radiopharmaceuticals. An orthogonal-targeting strategy and a contact-facilitated nanomicelle technology enabled highly selective delivery and co-localization of titanocene and radiolabelled fluorodeoxyglucose in disseminated multiple myeloma cells. Selective ablation of the cancer cells was achieved without significant off-target toxicity to the resident stem cells. Genomic, proteomic and multimodal imaging analyses revealed that the downregulation of CD49d, one of the dimeric protein targets of the nanomicelles, caused therapy resistance in small clusters of cancer cells. Similar treatment of a highly metastatic breast cancer model using human serum albumin-titanocene formulation significantly inhibited cancer growth. This strategy expands the use of phototherapy for treating previously inaccessible metastatic disease.

Monocytes Differentiate to Immune Suppressive Precursors of Metastasis-Associated Macrophages in Mouse Models of Metastatic Breast Cancer.

Metastasis-associated macrophages (MAMs) play pivotal roles in breast cancer metastasis by promoting extravasation and survival of metastasizing cancer cells. In a metastatic breast cancer mouse model, we previously reported that circulating classical monocytes (C-MOs) preferentially migrated into the tumor-challenged lung where they differentiated into MAMs. However, the fate and characteristics of C-MOs in the metastatic site has not been defined. In this study, we identified that adoptively transferred C-MOs (F4/80lowCD11b+Ly6C+) differentiated into a distinct myeloid cell population that is characterized as F4/80highCD11bhighLy6Chigh and gives rise to MAMs (F4/80lowCD11bhighLy6Clow) within 18 h after migration into the metastatic lung. In mouse models of breast cancer, the CD11bhighLy6Chigh MAM precursor cells (MAMPCs) were commonly found in the metastatic lung, and their accumulation was increased during metastatic tumor growth. The morphology and gene expression profile of MAMPCs were distinct from C-MOs and had greater similarity to MAMs. For example MAMPCs expressed mature macrophage markers such as CD14, CD36, CD64, and CD206 at comparable levels with MAMs, suggesting that MAMPCs have committed to a macrophage lineage in the tumor microenvironment. MAMPCs also expressed higher levels of Arg1, Hmox1, and Stab1 than C-MOs to a comparable level to MAMs. Expression of these MAM-associated genes in MAMPCs was reduced by genetic deletion of colony-stimulating factor 1 receptor (CSF1R). On the other hand, transient CSF1R blockade did not reduce the number of MAMPCs in the metastatic site, suggesting that CSF1 signaling is active in MAMPCs but is not required for their accumulation. Functionally MAMPCs suppressed the cytotoxicity of activated CD8+ T cells in vitro in part through superoxide production. Overall, our results indicate that immediately following migration into the metastatic tumors C-MOs differentiate into immunosuppressive cells that have characteristics of monocytic myeloid-derived suppressor cell phenotype and might be targeted to enhance efficacy of immunotherapy for metastatic breast cancer.

EDHSS206, an Exquisitely Selective Inhibitor of TAK1, Targets Pro Survival TNFFFDependent Signaling, Inducing Apoptosis in Rheumatoid Arthritis and Breast Cancer Models

Tumor necrosis factor alpha (TNFα) administration has shown limited therapeutic utility in cancer and inflammatory disorders due to dose‐limiting side effects. We propose an alternative approach to targeting TNFα‐mediated diseases by selectively inducing the TNFα apoptotic response in TNFα‐rich microenvironments. TAK1 acts as a key mediator between survival and cell death in TNFα signaling. We report a novel potent and exquisitely selective TAK1 inhibitor (EDHS‐206, IC50 9.5nM) that induces apoptosis in a TNFα‐dependent manner in models of metastatic breast cancer and rheumatoid arthritis. Co‐crystallization studies demonstrated that EDHS‐206 inhibits the kinase in a DFG‐in conformation. Kinetic analysis of EDHS‐206‐TAK1 interactions revealed a substrate‐like intermolecular autophosphorylation mechanism for TAK1 activation, during which EDHS‐206 delays the proceedings of the rate‐limiting step. EDHS‐206 represents an attractive starting point for the development of a novel generation of inhibitors that greatly sensitize cells to TNFα‐induced cell death, potentially broadening the therapeutic efficacy of TNFα.

Soft and flexible poly(ethylene glycol) nanotubes for local drug delivery.

Nanotubes are emerging as promising materials for healthcare applications but the selection of clinically relevant starting materials for their synthesis remains largely unexplored. Here we present, for the first time, the synthesis of poly(ethylene glycol) (PEG) based nanotubes via the photopolymerization of poly(ethylene glycol) diacrylate and other diacrylate derivatives within the pores of anodized aluminum oxide templates. Template-assisted synthesis allowed the manufacture of a diverse set of polymeric nanotubes with tunable physical characteristics including diameter (∼200-400 nm) and stiffness (405-902 kPa). PEG nanotubes were subjected to cytotoxicty assessment in cell lines and primary stem cells and showed excellent cytocompatability (IC50 > 120 μg ml-1). Nanotubes were readily drug loaded but released the majority of the drug over 5 days. Direct administration of drug loaded nanotubes to human orthotopic breast tumors substantially reduced tumor growth and metastasis and outperformed i.v. administration at the equivalent dose. Overall, this nanotube templating platform is emerging as a facile route for the manufacture of poly(ethylene glycol) nanotubes.

HPMA-Copolymer Nanocarrier Targets Tumor-Associated Macrophages in Primary and Metastatic Breast Cancer.

Polymeric nanocarriers such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers deliver drugs to solid tumors and avoid the systemic toxicity of conventional chemotherapy. Because HPMA copolymers can target sites of inflammation and accumulate within innate immune cells, we hypothesized that HPMA copolymers could target tumor-associated macrophages (TAM) in both primary and metastatic tumor microenvironments. We verified this hypothesis, first in preliminary experiments with isolated bone marrow macrophage cultures in vitro and subsequently in a spontaneously metastatic murine breast cancer model generated from a well-established, cytogenetically characterized 4T1 breast cancer cell line. Using our standardized experimental conditions, we detected primary orthotopic tumor growth at 7 days and metastatic tumors at 28 days after orthotopic transplantation of 4T1 cells into the mammary fat pad. We investigated the uptake of HPMA copolymer conjugated with Alexa Fluor 647 and folic acid (P-Alexa647-FA) and HPMA copolymer conjugated with IRDye 800CW (P-IRDye), following their retroorbital injection into the primary and metastatic tumor-bearing mice. A significant uptake of P-IRDye was observed at all primary and metastatic tumor sites in these mice, and the P-Alexa647-FA signal was found specifically within CD11b+ TAMs costained with pan-macrophage marker CD68. These findings demonstrate, for the first time, a novel capacity of a P-Alexa647-FA conjugate to colocalize to CD11b+CD68+ TAMs in both primary and metastatic breast tumors. This underscores the potential of this HPMA nanocarrier to deliver functional therapeutics that specifically target tumor-promoting macrophage activation and/or polarization during tumor development. Mol Cancer Ther; 16(12); 2701-10. ©2017 AACR.

Natural Killer T-cell Immunotherapy in Combination with Chemotherapy-Induced Immunogenic Cell Death Targets Metastatic Breast Cancer.

Natural killer T (NKT) cells are glycolipid-reactive lymphocytes that promote cancer control. In previous studies, NKT-cell activation improved survival and antitumor immunity in a postsurgical mouse model of metastatic breast cancer. Herein, we investigated whether NKT-cell activation could be combined with chemotherapeutic agents to augment therapeutic outcomes. Gemcitabine and cyclophosphamide analogues enhanced the potential immunogenicity of 4T1 mammary carcinoma cells by increasing the expression of antigen-presenting molecules (MHC-I, MHC-II, and CD1d) and promoting exposure or release of immunogenic cell death markers (calreticulin, HMGB1, and ATP). In 4T1 primary tumor and postsurgical metastasis models, BALB/c mice were treated with cyclophosphamide or gemcitabine. NKT cells were then activated by transfer of dendritic cells loaded with the glycolipid antigen α-galactosylceramide (α-GalCer). Chemotherapeutic treatments did not impact NKT-cell activation but enhanced recruitment into primary tumors. Cyclophosphamide, gemcitabine, or α-GalCer-loaded dendritic cell monotherapies decreased tumor growth in the primary tumor model and reduced metastatic burden and prolonged survival in the metastasis model. Combining chemotherapeutics with NKT-cell activation therapy significantly enhanced survival, with surviving mice exhibiting attenuated tumor growth following a second tumor challenge. The frequency of myeloid-derived suppressor cells was reduced by gemcitabine, cyclophosphamide, or α-GalCer-loaded dendritic cell treatments; cyclophosphamide also reduced the frequency of regulatory T cells. Individual treatments increased immune cell activation, cytokine polarization, and cytotoxic responses, although these readouts were not enhanced further by combining therapies. These findings demonstrate that NKT-cell activation therapy can be combined with gemcitabine or cyclophosphamide to target tumor burden and enhance protection against tumor recurrence. Cancer Immunol Res; 5(12); 1086-97. ©2017 AACR.

Efficacy in Treating Lung Metastasis of Invasive Breast Cancer with Functional Vincristine Plus Dasatinib Liposomes

Background: The metastasis of breast cancer is the leading cause of death, while lung metastasis is a major clinical phenomenon in patients with invasive breast cancer. The current treatment option comprising surgery, radiation, and standard chemotherapy cannot achieve a satisfactory effect on the treatment of lung metastasis of breast cancer. In this study, we report the potential of preventing lung metastasis of invasive breast cancer using the newly developed functional vincristine plus dasatinib liposomes. Methods: The investigations were performed on invasive breast cancer MDA-MB-231 cells in vitro and in lung metastatic model of invasive breast cancer MDA-MB-231 cells in nude mice. Results: The functional drug liposomes were able to induce cell cycle arrest at G2/M phase, induce apoptosis, inhibit adhesion, migration, and invasion of breast cancer cells in vitro, and prevent lung metastasis of breast cancer in nude mice. Conclusion: These findings indicate a potential clinical use of functional vincristine plus dasatinib liposomes for treating metastatic breast cancer.