Hayley J. Paholak

Activation of an IL6 Inflammatory Loop Mediates Trastuzumab Resistance in HER2+ Breast Cancer by Expanding the Cancer Stem Cell Population

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance.

Highly crystallized iron oxide nanoparticles as effective and biodegradable mediators for photothermal cancer therapy

We report that highly crystallized iron oxide nanoparticles (HCIONPs) made by thermal decomposition and further coating with a polysiloxane-containing copolymer can be used as effective mediators for photothermal therapy. Irradiation of a HCIONP solution containing 0.5 mg mL-1 Fe, for instance, with an 885 nm diode laser at a power of 2.5 W cm-2, induces a temperature increase of 33 °C from room temperature, while water produced only a ∼3 °C increase as the control. In vivo studies are further evaluated for effective photothermal therapy using the as-prepared HCIONPs. Benefiting from the great antibiofouling property of the polymer coating and minimized hydrodynamic size (whole particle size: 24 nm), the nanoparticles intravenously administered to SUM-159 tumor-bearing mice can effectively accumulate within the tumor tissue (5.3% of injection dose) through the enhanced permeability and retention effect. After applying the same laser conditions to irradiate the tumors, complete tumor regression is observed within three weeks without disease relapse over the course of three months. Conversely, control mice exhibit continuous tumor growth leading to animal mortality within four weeks. To better understand the photothermal effect of HCIONPs and potentially improve their photothermal efficiency, we compare their photothermal effect and crystal structures with commercially available magnetic nanoparticles. Our data show that after applying the same laser to commercially available magnetic nanoparticles from FeREX at the same iron concentration, the temperature is only increased by 7.4 °C. We further use synchrotron-XRD and high-resolution TEM to compare the crystal structures of both magnetic nanoparticles. The data show that both magnetic nanoparticles are Fe3O4 but as-prepared HCIONPs are highly crystalline and have preferred lattice plane orientations, which may be the cause of their enhanced photothermal efficiency. Taken together, these data suggest that HCIONPs, with unique lattice orientations and small size as well as antifouling coating, can be used as promising mediators for photothermal cancer therapy.

Applications of Human Pharmacokinetic Prediction in First-in-Human Dose Estimation

Quantitative estimations of first-in-human (FIH) doses are critical for phase I clinical trials in drug development. Human pharmacokinetic (PK) prediction methods have been developed to project the human clearance (CL) and bioavailability with reasonable accuracy, which facilitates estimation of a safe yet efficacious FIH dose. However, the FIH dose estimation is still very challenging and complex. The aim of this article is to review the common approaches for FIH dose estimation with an emphasis on PK-guided estimation. We discuss 5 methods for FIH dose estimation, 17 approaches for the prediction of human CL, 6 methods for the prediction of bioavailability, and 3 tools for the prediction of PK profiles. This review may serve as a practical protocol for PK- or pharmacokinetic/pharmacodynamic-guided estimation of the FIH dose.

Noninvasive Fluorescence Resonance Energy Transfer Imaging of in Vivo Premature Drug Release from Polymeric Nanoparticles

Understanding in vivo drug release kinetics is critical for the development of nanoparticle-based delivery systems. In this study, we developed a fluorescence resonance energy transfer (FRET) imaging approach to noninvasively monitor in vitro and in vivo cargo release from polymeric nanoparticles. The FRET donor dye (DiO or DiD) and acceptor dye (DiI or DiR) were individually encapsulated into poly(ethylene oxide)-b-polystyrene (PEO-PS) nanoparticles. When DiO (donor) nanoparticles and DiI (acceptor) nanoparticles were coincubated with cancer cells for 2 h, increased FRET signals were observed from cell membranes, suggesting rapid release of DiO and DiI to cell membranes. Similarly, increased FRET ratios were detected in nude mice after intravenous coadministration of DiD (donor) nanoparticles and DiR (acceptor) nanoparticles. In contrast, another group of nude mice i.v. administrated with DiD/DiR coloaded nanoparticles showed decreased FRET ratios. Based on the difference in FRET ratios between the two groups, in vivo DiD/DiR release half-life from PEO-PS nanoparticles was determined to be 9.2 min. In addition, it was observed that the presence of cell membranes facilitated burst release of lipophilic cargos while incorporation of oleic acid-coated iron oxide into PEO-PS nanoparticles slowed the release of DiD/DiR to cell membranes. The developed in vitro and in vivo FRET imaging techniques can be used to screening stable nanoformulations for lipophilic drug delivery.

Intracellular dissociation of a polymer coating from nanoparticles

Polymer-coated nanoparticles are widely used for drug delivery in cancer therapy. However, it is not clear whether the polymer coating is disrupted in the lipid bilayer or intracellular space. Our current work suggests that the polymer coating of inorganic nanoparticles is disrupted after internalization by cancer cells. Single dispersed red quantum dots (QDs) labeled with 5-carboxyfluorescein (5-FAM) (green) (diameter = 28 nm) were incubated with cancer cells (PC-3) and imaged via fluorescence microscopy. Initially the 5-FAM-labeled polymer coating was attached to the QD and its green fluorescence was quenched when the nanoparticles were internalized after 4 h incubation, but the 5-FAM-labeled polymer became separated from the QD once inside the lysosomes of cells and its fluorescence becomes visible after 8 h. The fluorescence ratio (5-FAM/QDs) was increased 29-fold after 8 h incubation compared to 2 h. The fluorescence quenching effect of PEG-5-FAM after conjugation in solution (quenched by 44%) was compared to free poly(ethylene glycol)-5-FAM (PEG-5-FAM) mixing with QDs, which only exhibited slight (6.9%) quenching of 5-FAM. In addition, the intracellular dissociation of polymer coating from QD loaded micelles (diameter = 300 nm) was also observed. Furthermore, amphiphilic polymer labeled with the hydrophobic dye 6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)hexanoic acid (BODIPY® TMR) (red) was applied to encapsulate hydrophobic iron oxide nanoparticles (IONPs). The BODIPY dye was quenched by both the encapsulated IONPs and the hydrophobic region inside the micelles, while an 8-fold fluorescence enhancement was observed after polymeric micelle dissociation. Our in vitro results also reveal the polymeric dissociation after internalization by cancer cells as the dye signal becomes detectable after 24 h incubation. These results suggest that the polymer coating is stable in the lipid bilayer and becomes dissociated from nanoparticles in the lysosome of cancer cells. These data will provide guidance for intracellular drug delivery using polymer coated nanoparticles.Graphical abstract

Facile Fabrication of Near-Infrared-Resonant and Magnetic Resonance Imaging-Capable Nanomediators for Photothermal Therapy

Although many techniques exist for fabricating near-infrared (NIR)-resonant and magnetic resonance imaging (MRI)-capable nanomediators for photothermal cancer therapy, preparing them in an efficient and scalable process remains a significant challenge. In this report, we exploit one-step siloxane chemistry to facilely conjugate NIR-absorbing satellites onto a well-developed polysiloxane-containing polymer-coated iron oxide nanoparticle (IONP) core to generate dual functional core-satellite nanomediators for photothermal therapy. An advantage of this nanocomposite design is the variety of potential satellites that can be simply attached to impart NIR resonance, which we demonstrate using NIR-resonant gold sulfide nanoparticles (Au2SNPs) and the NIR dye IR820 as two example satellites. The core-satellite nanomediators are fully characterized by using absorption spectra, dynamic light scattering, ζ potential measurements, and transmission electron microscopy. The enhanced photothermal effect under the irradiation of NIR laser light is identified through in vitro solutions and in vivo mice studies. The MRI capabilities as contrast agents are demonstrated in mice. Our data suggest that polysiloxane-containing polymer-coated IONPs can be used as a versatile platform to build such dual functional nanomediators for translatable, MRI-guided photothermal cancer therapy.

Thiol-reactive amphiphilic block copolymer for coating gold nanoparticles with neutral and functionable surfaces

Nanoparticles designed for biomedical applications are often coated with polymers containing reactive functional groups, such as -COOH and -NH2, to conjugate targeting ligands or drugs. However, introducing highly charged surfaces promotes binding of the nanoparticles to biomolecules in biological systems through ionic interactions, causing the nanoparticles to aggregate in biological environments and consequently undergo strong non-specific binding to off-target cells and tissues. Developing a unique polymer with neutral surfaces that can be further functionalized directly would be critical to develop suitable nanomaterials for nanomedicine. Here, we report a thiol-reactive amphiphilic block copolymer poly(ethylene oxide)-block-poly(pyridyldisulfide ethylmeth acrylate) (PEO-b-PPDSM) for coating gold nanoparticles (AuNPs). The resultant polymer-coated AuNPs have almost neutral surfaces with slightly negative zeta potentials from -10 to 0 mV over a wide pH range from 2 to 12. Although the zeta potential is close to zero we show that the PEO-b-PPDSM copolymer-coated AuNPs have both good stability in various physiological conditions and reduced non-specific adsorption of proteins/biomolecules. Because of the multiple pyridyldisulfide groups on the PPDSM block, these individually dispersed nanocomplexes with an overall hydrodynamic size around 43.8 nm can be directly functionalized via disulfide-thiol exchange chemistry.

Abstract 4076: Combination of docetaxel with sulforaphane synergistically inhibits triple negative breast cancer and cancer stem cells

Triple negative breast cancer (TNBC) (ER-, PR-, Her2-), constituting 10-20% of all breast cancers, is a heterogeneous disease with limited treatment options and poor prognosis. TNBCs exhibit rapid progression with the duration of response to first line palliative chemotherapy typically less than 12 weeks, and overall five year survival of patients with metastatic TNBC of 22%. The cancer stem cell (CSC) model provides an attractive explanation for relapse of TNBC after primary therapy since these cells demonstrate resistance to conventional chemotherapy. CSCs which survive primary treatments, such as docetaxel, may self-renew and differentiate into the heterogeneous tumor bulk resulting in local recurrence and distant metastasis. Docetaxel has been demonstrated to not only fail to eliminate CSCs but expands this population in preclinical models. Further, docetaxel increases circulating IL-6 in patients following therapy, a cytokine reported to expand breast CSCs. Therefore, we sought to combine docetaxel with a small molecule CSC inhibitor capable of reducing IL-6 production (sulforaphane, SF) for the effective treatment of TNBCs. Our results in vitro demonstrate that docetaxel treatment (5 nM) increases the proportion of CSCs in TNBC cell lines (SUM149 and SUM159) as evident by flow cytometry analysis using the ALDEFLUOR assay (70.6±22.0%) and cells which are CD44+/CD24-/EpCAM+ (2.9 fold). Mammosphere formation assay reveals 1 nM docetaxel increases secondary sphere formation rate by 75.8±29%. As determined by ELISA, 5 nM docetaxel treatment for 72 hours induces 3.5 fold increase in IL-6 production. Conversely, SF (2.5 μM) selectively reduces the proportion of ALDEFLUOR positive cells (51.5±15.0%) and mammosphere formation (39.2±3.8%) while reducing IL-6 (55.6±5.0%) through regulation of NF-kB activity. In combination docetaxel and SF synergize to effectively reduce bulk cell line proliferation (combination index range 1-0.093). Further, SF prevents docetaxel mediated CSC expansion and IL-6 production. Using a mouse xenograft model docetaxel (10 mg/kg weekly) reduces tumor growth of established tumors by 83.2±6.0% whereas SF (50 mg/kg daily) inhibits primary tumor growth by 37.4±14.6%. In addition, secondary reimplantation assays with limiting dilution analysis reveals docetaxel increases the frequency of the tumor initiating CSCs (1/1514 control cells vs 1/330 docetaxel treated cells) while SF reduces the frequency to 1/3181 cells. In vivo, the combination of docetaxel and SF exhibits a greater reduction in primary tumor volume (92.5±2.1% reduction relative to control), and synergistically inhibit the CSC population (1 in 4245 cells). These results suggest that SF mediated inhibition of breast CSCs and IL-6 provide a scientific rationale for using this agent in combination with docetaxel for TNBC. Citation Format: Joseph P. Burnett, Ronack B. Shah, Hayley J. Paholak, Sean P. McDermott, Yasuhiro Tsume, Max W. Wicha, Duxin Sun. Combination of docetaxel with sulforaphane synergistically inhibits triple negative breast cancer and cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4076. doi:10.1158/1538-7445.AM2015-4076

Abstract 3681: Facile fabrication of MRI-capable and NIR-resonant core-satellite nanomediators for photothermal therapy

Although many techniques exist for fabricating near-infrared (NIR)-resonant and magnetic resonance imaging (MRI)-capable nanomediators for photothermal cancer therapy, preparing them in an efficient and scalable process remains a significant challenge. In this report, we develop two facile methods to fabricate both NIR-resonant and MRI-capable nanomediators utilizing well developed polysiloxane-containing polymer-coated iron oxide nanoparticles (IONPs). One of the methods is to attach multiple NIR-resonant nanoparticles onto a thiol-modified IONP core through one-step mixing with gold sulfide nanoparticles (Au2S NPs) to fabricate a core-satellite nanomediator. The other strategy is to covalently attach a NIR organic dye onto intact polysiloxane-containing polymer-coated IONPs through facile mixing with siloxane-modified IR820. The successful decoration with either Au2S NPs or IR820 is demonstrated for the resultant nanomediators by absorption spectra and transmission electron microscopy (TEM) images. It is estimated that 10 Au2S NPs and 2×104 dye molecules (loading capacity: 4.5 mg IR820/mg Fe) are attached onto the polymer-coated IONPs, respectively. The hydrodynamic sizes of these novel multifunctional nanomediators are ∼57.5 nm for IONP-Au2S NP and 28.2 nm for IONP-IR820, which are much smaller than traditional gold nanoshell-based designs. The photothermal efficiency of the IONP-Au2S NP and IONP-IR820 nanocomposite solutions under the irradiation of 885 nm NIR laser light is significantly enhanced compared to IONPs alone. The enhancement in photothermal efficiency by the newly developed nanomediators is also investigated in nude mice bearing SUM-159 tumor xenografts. The biodistribution data measured by inductively coupled plasma optical emission spectrometry (ICP-OES) reveal that ∼1% injection dose (ID) of IONP-Au2S NPs and ∼9% ID of IONP-IR820 accumulate into tumor tissue 24 h post intravenous injection at a dose of 20 mg Fe/Kg mouse body weight. The average tumor surface temperature recorded by an infrared camera is increased by 10.8 ± 1.2 °C for the mice administered with IONP-Au2S NPs and 25.7 ± 3.6 °C for the group injected with IONP-IR820, compared to 4.8 ± 0.5 °C for the PBS-injected control mice after laser irradiation with a power of 0.5 W for 10 min. Furthermore, our data reveal that the fabrication will not compromise the IONP core capability as an MRI contrast agent. Taken together, we demonstrate a facile technique to generate NIR-resonant and MRI-capable nanomediators from a polysiloxane-containing polymer-coated IONP platform. The as-developed dual functional nanomediators hold great promise for translatable MRI-guided photothermal cancer therapy. Citation Format: Hongwei Chen, Xiaoqing Ren, Hayley Paholak, Joseph Burnett, Feng Ni, Duxin Sun. Facile fabrication of MRI-capable and NIR-resonant core-satellite nanomediators for photothermal therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3681. doi:10.1158/1538-7445.AM2015-3681

Abstract 2002: Disrupting immunosuppressive tumor microenvironment via stealthy nanobombs to enhance T-cell anticancer efficacy

Despite the fact that the immunosuppressive tumor microenvironment (ISTME) has long been recognized as a major hurdle that significantly limits the anticancer efficacy of various therapeutic strategies, including T-cell-based immunotherapy, there is currently no effective way to address this challenge. In this work, we prove that sequential photothermal therapy (PTT), mediated with stealthy laser-triggerable nanobombs composed of iron oxide nanoparticles (IONPs) following systemic delivery, can remove both pre-resident and also newly triggered immunosuppressor cells in established tumors to enhance the efficacy of T-cell anticancer therapy. Using flow cytometry to study various T-cell populations in tumor tissues 48 h post PTT in a 4T1 mouse model, our data suggest that IONP-mediated PTT could significantly reduce the CD4+FoxP3+ regulatory T-cell population. Our data also suggest that CD8+ cytotoxic T-cell populations in PTT-treated tumor tissues return to those of non-treated tumors, and are believed to be newly activated at the tumor site or recruited from periphery blood following PTT. Our data further suggest that PTT twice spaced by a 24 h interval has the best anticancer efficacy, compared to PTT once or three times at 24 h increments. This enhanced anticancer effect from the sequential IONP-mediated PTT (twice) is believed to be due to an enhanced ability to eliminate triggered immunosuppressor cells that respond faster than cytotoxic CD8+ T-cells. Furthermore, our Luminex analysis of chemokine serum concentrations indicates that IONP-mediated PTT can decrease suppressor-cell-attractive protein secretion, such as G-CSF. In addition, our immunohistochemistry data from tumor tissue staining indicate that IONP-mediated PTT down-regulates tumor cell PD-L1 expression to disrupt tumor-cell-mediated immunosuppression. The capability of IONP-mediated PTT to disrupt ISTME can significantly enhance T-cell anticancer efficacy toward both treated established tumors and distal tumor cells. When combining IONP-mediated PTT with anti-CTLA-4 therapy to block CD8+ T-cell inhibition, our data suggest that combination treatment significantly inhibits tumor growth (p Citation Format: Hongwei Chen, Hayley J. Paholak, Xin Luan, Joseph P. Burnett, Nicholas O. Stevers, Kanokwan Sansanaphongpricha, Duxin Sun. Disrupting immunosuppressive tumor microenvironment via stealthy nanobombs to enhance T-cell anticancer efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2002. doi:10.1158/1538-7445.AM2017-2002