Randomised phase II trial of gemcitabine and nab-paclitaxel with necuparanib or placebo in untreated metastatic pancreas ductal adenocarcinoma.

BACKGROUND: Necuparanib, a rationally engineered low-molecular-weight heparin, combined with gemcitabine/nab-paclitaxel showed an encouraging safety and oncologic signal in a phase Ib trial. This randomised multicentre phase II trial evaluates the addition of necuparanib or placebo to gemcitabine/nab-paclitaxel in untreated metastatic pancreatic ductal adenocarcinoma (PDAC). PATIENTS AND METHODS: Eligibility included 18 years, histologically or cytologically confirmed metastatic PDAC, measurable disease and Eastern Co-Operative Oncology Group performance status of 0-1. Patients were randomly assigned to necuparanib (5 mg/kg subcutaneous injection once daily) or placebo (subcutaneous injection once daily) and gemcitabine/nab-paclitaxel on days 1, 8 and 15 of 28-day cycles. The primary end-point was median overall survival (OS), and secondary end-points included median progression-free survival, response rates and safety. RESULTS: One-hundred ten patients were randomised, 62 to necuparanib arm and 58 to placebo arm. The futility boundary was crossed at a planned interim analysis, and the study was terminated by the Data Safety Monitoring Board. The median OS was 10.71 months (95% confidence interval [CI]: 7.95-11.96) for necuparanib arm and 9.99 months (95% CI: 7.85-12.85) for placebo arm (hazard ratio: 1.12, 95% CI: 0.66-1.89, P-value: 0.671). The necuparanib arm had a higher incidence of haematologic toxicity relative to placebo patients (83% and 70%). CONCLUSION: The addition of necuparanib to standard of care treatment for advanced PDAC did not improve OS. Safety was acceptable. No further development of necuparanib is planned although targeting the coagulation cascade pathway remains relevant in PDAC. NCT01621243.

Necuparanib, A Multitargeting Heparan Sulfate Mimetic, Targets Tumor and Stromal Compartments in Pancreatic Cancer.

Pancreatic cancer has an abysmal 5-year survival rate of 8%, making it a deadly disease with a need for novel therapies. Here we describe a multitargeting heparin-based mimetic, necuparanib, and its antitumor activity in both in vitro and in vivo models of pancreatic cancer. Necuparanib reduced tumor cell proliferation and invasion in a three-dimensional (3D) culture model; in vivo, it extended survival and reduced metastasis. Furthermore, proteomic analysis demonstrated that necuparanib altered the expression levels of multiple proteins involved in cancer-driving pathways including organ development, angiogenesis, proliferation, genomic stability, cellular energetics, and invasion and metastasis. One protein family known to be involved in invasion and metastasis and altered by necuparanib treatment was the matrix metalloprotease (MMP) family. Necuparanib reduced metalloproteinase 1 (MMP1) and increased tissue inhibitor of metalloproteinase 3 (TIMP3) protein levels and was found to increase RNA expression of TIMP3. MMP enzymatic activity was also found to be reduced in the 3D model. Finally, we confirmed necuparanib's in vivo activity by analyzing plasma samples of patients enrolled in a phase I/II study in patients with metastatic pancreatic cancer; treatment with necuparanib plus standard of care significantly increased TIMP3 plasma protein levels. Together, these results demonstrate necuparanib acts as a broad multitargeting therapeutic with in vitro and in vivo anti-invasive and antimetastatic activity.

Safety, Pharmacokinetics, Pharmacodynamics, and Antitumor Activity of Necuparanib Combined with Nab-Paclitaxel and Gemcitabine in Patients with Metastatic Pancreatic Cancer: Phase I Results.

LESSONS LEARNED: Despite the compelling preclinical rationale of evaluating the genetically engineered heparin derivative, necuparanib, combined with standard therapy in metastatic pancreas adenocarcinoma, the results were ultimately disappointing.Safety was documented, although dose escalation was limited by the number of subcutaneous injections, the potential for skin toxicity (cellulitis), and low-level anticoagulant effect. Nonetheless, the hypothesis of targeting prothrombotic pathways in pancreas adenocarcinoma remains compelling. BACKGROUND: Necuparanib is derived from unfractionated heparin and engineered for reduced anticoagulant activity while preserving known heparin-associated antitumor properties. This trial assessed the safety, pharmacokinetics (PK), pharmacodynamics, and initial efficacy of necuparanib combined with gemcitabine ± nab-paclitaxel in patients with metastatic pancreatic cancer. METHODS: Patients received escalating daily subcutaneous doses of necuparanib plus 1,000 mg/m2 gemcitabine (days 1, 8, 15, and every 28 days). The protocol was amended to include 125 mg/m2 nab-paclitaxel after two cohorts (following release of the phase III MPACT data). The necuparanib starting dose was 0.5 mg/kg, with escalation via a modified 3 + 3 design until the maximum tolerated dose (MTD) was determined. RESULTS: Thirty-nine patients were enrolled into seven cohorts (necuparanib 0.5, 1 mg/kg + gemcitabine; necuparanib 1, 2, 4, 6, and 5 mg/kg + nab-paclitaxel + gemcitabine). The most common adverse events were anemia (56%), fatigue (51%), neutropenia (51%), leukopenia (41%), and thrombocytopenia (41%). No deaths and two serious adverse events were potentially related to necuparanib. Measurable levels of necuparanib were seen starting at the 2 mg/kg dose. Of 24 patients who received at least one dose of necuparanib + nab-paclitaxel + gemcitabine, 9 (38%) achieved a partial response and 6 (25%) achieved stable disease (63% disease control rate). Given a cellulitis event and mild activated partial thromboplastin time increases at 6 mg/kg, the 5 mg/kg dose was considered the MTD and selected for further assessment in phase II. CONCLUSION: Acceptable safety and encouraging signals of activity in patients with metastatic pancreatic cancer receiving necuparanib, nab-paclitaxel, and gemcitabine were demonstrated.

Control of cancer formation by intrinsic genetic noise and microenvironmental cues.

Differentiation therapies that induce malignant cells to stop growing and revert to normal tissue-specific differentiated cell types are successful in the treatment of a few specific haematological tumours. However, this approach has not been widely applied to solid tumours because their developmental origins are less well understood. Recent advances suggest that understanding tumour cell plasticity and how intrinsic factors (such as genetic noise and microenvironmental signals, including physical cues from the extracellular matrix) govern cell state switches will help in the development of clinically relevant differentiation therapies for solid cancers.

Silencing HoxA1 by intraductal injection of siRNA lipidoid nanoparticles prevents mammary tumor progression in mice.

With advances in screening, the incidence of detection of premalignant breast lesions has increased in recent decades; however, treatment options remain limited to surveillance or surgical removal by lumpectomy or mastectomy. We hypothesized that disease progression could be blocked by RNA interference (RNAi) therapy and set out to develop a targeted therapeutic delivery strategy. Using computational gene network modeling, we identified HoxA1 as a putative driver of early mammary cancer progression in transgenic C3(1)-SV40TAg mice. Silencing this gene in cultured mouse or human mammary tumor spheroids resulted in increased acinar lumen formation, reduced tumor cell proliferation, and restoration of normal epithelial polarization. When the HoxA1 gene was silenced in vivo via intraductal delivery of nanoparticle-formulated small interfering RNA (siRNA) through the nipple of transgenic mice with early-stage disease, mammary epithelial cell proliferation rates were suppressed, loss of estrogen and progesterone receptor expression was prevented, and tumor incidence was reduced by 75%. This approach that leverages new advances in systems biology and nanotechnology offers a novel noninvasive strategy to block breast cancer progression through targeted silencing of critical genes directly within the mammary epithelium.

Intraductal injection for localized drug delivery to the mouse mammary gland.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

How changes in extracellular matrix mechanics and gene expression variability might combine to drive cancer progression.

Changes in extracellular matrix (ECM) structure or mechanics can actively drive cancer progression; however, the underlying mechanism remains unknown. Here we explore whether this process could be mediated by changes in cell shape that lead to increases in genetic noise, given that both factors have been independently shown to alter gene expression and induce cell fate switching. We do this using a computer simulation model that explores the impact of physical changes in the tissue microenvironment under conditions in which physical deformation of cells increases gene expression variability among genetically identical cells. The model reveals that cancerous tissue growth can be driven by physical changes in the microenvironment: when increases in cell shape variability due to growth-dependent increases in cell packing density enhance gene expression variation, heterogeneous autonomous growth and further structural disorganization can result, thereby driving cancer progression via positive feedback. The model parameters that led to this prediction are consistent with experimental measurements of mammary tissues that spontaneously undergo cancer progression in transgenic C3(1)-SV40Tag female mice, which exhibit enhanced stiffness of mammary ducts, as well as progressive increases in variability of cell-cell relations and associated cell shape changes. These results demonstrate the potential for physical changes in the tissue microenvironment (e.g., altered ECM mechanics) to induce a cancerous phenotype or accelerate cancer progression in a clonal population through local changes in cell geometry and increased phenotypic variability, even in the absence of gene mutation.

Breast cancer normalization induced by embryonic mesenchyme is mediated by extracellular matrix biglycan.

Some epithelial cancers can be induced to revert to quiescent differentiated tissue when combined with embryonic mesenchyme; however, the mechanism of this induction is unknown. Here we combine tissue engineering, developmental biology, biochemistry and proteomics approaches to attack this problem. Using a synthetic reconstitution system, we show that co-culture of breast cancer cells with embryonic mesenchyme from early stage (E12.5-13.5) mammary glands decreases tumor cell proliferation while stimulating acinus differentiation, whereas cancer-associated fibroblasts (CAFs) fail to produce these normalizing effects. When insoluble extracellular matrices (ECMs) were isolated from cultured early stage (E12.5-13.5) embryonic mammary mesenchyme cells or E10 tooth mesenchyme and recombined with mammary tumor cells, they were found to be sufficient to induce breast cancer normalization including enhanced expression of estrogen receptor-α (ER-α). In contrast, ECM from later stage (E14.5) mammary mesenchyme and conditioned medium isolated from mesenchymal cell cultures were ineffective. Importantly, when the inductive ECMs produced by early stage embryonic mammary mesenchyme were scraped from dishes and injected into fast-growing breast tumors in mice, they significantly inhibited cancer expansion. Proteomics analysis of the detergent insoluble ECM material revealed several matrix components that were preferentially expressed in the embryonic ECMs. Analysis of two of these molecules previously implicated in cancer regulation--biglycan and tenascin C--revealed that addition of biglyan can mimic the tumor normalization response, and that siRNA knockdown of its expression in cultured embryonic mesenchyme results in loss of the ECM's inductive activity. These studies confirm that embryonic mesenchyme retains the ability to induce partial breast cancer reversion, and that its inductive capability resides at least in part in the ECM protein biglycan that it produces.

A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells.

Here we describe a combined microfluidic-micromagnetic cell separation device that has been developed to isolate, detect and culture circulating tumor cells (CTCs) from whole blood, and demonstrate its utility using blood from mammary cancer-bearing mice. The device was fabricated from polydimethylsiloxane and contains a microfluidic architecture with a main channel and redundant 'double collection' channel lined by two rows of dead-end side chambers for tumor cell collection. The microdevice design was optimized using computational simulation to determine dimensions, magnetic forces and flow rates for cell isolation using epithelial cell adhesion molecule (EpCAM) antibody-coated magnetic microbeads (2.8 µm diameter). Using this device, isolation efficiencies increased in a linear manner and reached efficiencies close to 90% when only 2 to 80 breast cancer cells were spiked into a small volume (1.0 mL) of blood taken from wild type mice. The high sensitivity visualization capabilities of the device also allowed detection of a single cell within one of its dead-end side chambers. When blood was removed from FVB C3(1)-SV40 T-antigen mammary tumor-bearing transgenic mice at different stages of tumor progression, cells isolated in the device using anti-EpCAM-beads and magnetically collected within the dead-end side chambers, also stained positive for pan-cytokeratin-FITC and DAPI, negative for CD45-PerCP, and expressed SV40 large T antigen, thus confirming their identity as CTCs. Using this isolation approach, we detected a time-dependent rise in the number of CTCs in blood of female transgenic mice, with a dramatic increase in the numbers of metastatic tumor cells appearing in the blood after 20 weeks when tumors transition to invasive carcinoma and exhibit increased growth of metastases in this model. Importantly, in contrast to previously described CTC isolation methods, breast tumor cells collected from a small volume of blood removed from a breast tumor-bearing animal remain viable and they can be easily removed from these devices and expanded in culture for additional analytical studies or potential drug sensitivity testing.

Dual regulation of breast tubulogenesis using extracellular matrix composition and stromal cells.

Epithelial-mesenchymal interactions during embryogenesis are critical in defining the phenotype of tissues and organs. The initial elongation of the mammary bud represents a central morphological event requiring extensive epithelial-mesenchymal crosstalk. The precise mechanism orchestrating this outgrowth is still unknown and mostly animal models have been relied upon to explore this process. Highly tunable three-dimensional (3D) culture models are a complementary approach to address the question of phenotypic determination. Here, we used a 3D in vitro culture to study the roles of stromal cells and extracellular matrix components during mammary tubulogenesis. Fibroblasts, adipocytes, and type I collagen actively participated in this process, whereas reconstituted basement membrane inhibited tubulogenesis by affecting collagen organization. We conclude that biochemical and biomechanical signals mediate the interaction between cells and matrix components and are necessary to induce tubulogenesis in vitro.

The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures.

BACKGROUND: Stromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D) in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination. METHODS: The human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks), the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square. RESULTS: Most of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures. CONCLUSIONS: Our results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to induce near-complete tumor phenotype reversion. These human breast 3D tissue morphogenesis models promise to become reliable tools for studying tissue interactions, therapeutic screening and drug target validation.

A novel 3D in vitro culture model to study stromal-epithelial interactions in the mammary gland.

Stromal-epithelial interactions mediate mammary gland development and the formation and progression of breast cancer. To study these interactions in vitro, the development of defined three-dimensional (3D) models is essential. In the present study, we have successfully developed novel 3D in vitro models that allow the formation of mammary gland structures closely resembling those found in vivo. Cocultures of a human mammary epithelial cell line MCF10A and human mammary fibroblasts obtained from reduction mammoplasties embedded in either a type I collagen or a mixed Matrigel-collagen matrix were carried out for up to 6 weeks. Histological and ultrastructural analysis confirmed the formation of ductal and alveolar structures. The importance of the stromal cells was apparent in both matrices; in the collagen gels the presence of reduction mammoplasty fibroblasts accelerated the initial formation of epithelial structures, and in the mixed Matrigel-collagen gels the presence of those fibroblasts was necessary for the formation of ductal structures. These models provide an excellent system to study tissue organization, epithelial morphogenesis, and breast carcinogenesis.