The SRC-family serves as a therapeutic target in triple negative breast cancer with acquired resistance to chemotherapy.

BACKGROUND: Resistance to chemotherapy, combined with heterogeneity among resistant tumors, represents a significant challenge in the clinical management of triple negative breast cancer (TNBC). By dissecting molecular pathways associated with treatment resistance, we sought to define patient sub-groups and actionable targets for next-line treatment. METHODS: Bulk RNA sequencing and reverse phase protein array profiling were performed on isogenic patient-derived xenografts (PDX) representing paclitaxel-sensitive and -resistant tumors. Pathways identified as upregulated in the resistant model were further explored as targets in PDX explants. Their clinical relevance was assessed in two distinct patient cohorts (NeoAva and MET500). RESULTS: Increased activity in signaling pathways involving SRC-family kinases (SFKs)- and MAPK/ERK was found in treatment resistant PDX, with targeted inhibitors being significantly more potent in resistant tumors. Up-regulation of SFKs- and MAPK/ERK-pathways was also detected in a sub-group of chemoresistant patients after neoadjuvant treatment. Furthermore, High SFK expression (of either SRC, FYN and/or YES1) was detected in metastatic lesions of TNBC patients with fast progressing disease (median disease-free interval 27 vs 105 months). CONCLUSIONS: Upregulation of SFK-signaling is found in a subset of chemoresistant tumors and is persistent in metastatic lesions. Based on pre-clinical results, these patients may respond favorably to treatment targeting SFKs.

Drug-Loaded Photosensitizer-Chitosan Nanoparticles for Combinatorial Chemo- and Photodynamic-Therapy of Cancer.

In this study we have developed biodegradable polymeric nanoparticles (NPs) containing the cytostatic drugs mertansine (MRT) or cabazitaxel (CBZ). The NPs are based on chitosan (CS) conjugate polymers synthesized with different amounts of the photosensitizer tetraphenylchlorin (TPC). These TPC-CS NPs have high loading capacity and strong drug retention due to π-π stacking interactions between the drugs and the aromatic photosensitizer groups of the polymers. CS polymers with 10% of the side chains containing TPC were found to be optimal in terms of drug loading capacity and NP stability. The TPC-CS NPs loaded with MRT or CBZ displayed higher cytotoxicity than the free form of these drugs in the breast cancer cell lines MDA-MB-231 and MDA-MB-468. Furthermore, light-induced photochemical activation of the NPs elicited a strong photodynamic therapy effect on these breast cancer cells. Biodistribution studies in mice showed that most of the TPC-CS NPs accumulated in liver and lungs, but they were also found to be localized in tumors derived from HCT-116 cells. These data suggest that the drug-loaded TPC-CS NPs have a potential in combinatory anticancer therapy and as contrast agents.

Tube formation in the first trimester placental trophoblast cells: Differential effects of angiogenic growth factors and fatty acids.

The study aims to investigate whether cytosolic fatty acid-binding protein-4 (FABP4) is involved in angiogenic growth factors- and fatty acid-induced tube formation in first trimester placental trophoblast cells, HTR8/SVneo. We determined the tube formation both at basal as well as stimulated levels in the absence and presence of inhibitors of FABP4 and VEGF signaling pathways. Basal level of tube formation was maximally reduced in the presence of 50 µM of FABP4 inhibitor compared with those by VEGF signaling pathway inhibitors (rapamycin, L-NAME, and p38 MAP kinase inhibitor). Whereas docosahexaenoic acid, 22:6n-3 (DHA)-, and VEGF-induced tube formation was maximally inhibited by p38 MAP kinase inhibitor (63.7 and 34.5%, respectively), however, leptin-induced tube formation was inhibited maximally by FABP4 inhibitor (50.7%). ANGPTL4 and oleic acid (OA)-induced tube formation was not blocked by any of these inhibitors. The FABP4 inhibitor inhibited cell growth stimulated by DHA, leptin, VEGF, and OA (P < 0.05) but was not affected by ANGPTL4. VEGF, leptin, and OA also increased FABP4 protein level in these cells, though the uptake of fatty acids by these cells was not affected by the presence of FABP4 inhibitor. Our data demonstrate that FABP4 may be involved in part in the basal level, and stimulated tube formation by VEGF, DHA, and leptin, whereas it has little or no effect in ANGPTL4- and OA-induced tube formation in these cells. Thus, FABP4 may play a differential role in fatty acids and angiogenic growth factors-mediated tube formation in the first trimester trophoblast cells in vitro.

Preclinical Efficacy of Cabazitaxel Loaded Poly(2-alkyl cyanoacrylate) Nanoparticle Variants.

Background: Biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs) are receiving increasing attention in anti-cancer nanomedicine development not only for targeted cancer chemotherapy, but also for modulation of the tumor microenvironment. We previously reported promising results with cabazitaxel (CBZ) loaded poly(2-ethylbutyl cyanoacrylate) NPs (PEBCA-CBZ NPs) in a patient derived xenograft (PDX) model of triple-negative breast cancer, and this was associated with a decrease in M2 macrophages. The present study aims at comparing two endotoxin-free PACA NP variants (PEBCA and poly(2-ethylhexyl cyanoacrylate); PEHCA), loaded with CBZ and test whether conjugation with folate would improve their effect. Methods: Cytotoxicity assays and cellular uptake of NPs by flow cytometry were performed in different breast cancer cells. Biodistribution and efficacy studies were performed in PDX models of breast cancer. Tumor associated immune cells were analyzed by multiparametric flow cytometry. Results: In vitro studies showed similar NP-induced cytotoxicity patterns despite difference in early NP internalization. On intravenous injection, the liver cleared the majority of NPs. Efficacy studies in the HBCx39 PDX model demonstrated an enhanced effect of drug-loaded PEBCA variants compared with free drug and PEHCA NPs. Furthermore, the folate conjugated PEBCA variant did not show any enhanced effects compared with the unconjugated counterpart which might be due to unfavorable orientation of folate on the NPs. Finally, analyses of the immune cell populations in tumors revealed that treatment with drug loaded PEBCA variants affected the myeloid cells, especially macrophages, contributing to an inflammatory, immune activated tumor microenvironment. Conclusion: We report for the first time, comparative efficacy of PEBCA and PEHCA NP variants in triple negative breast cancer models and show that CBZ-loaded PEBCA NPs exhibit a combined effect on tumor cells and on the tumor associated myeloid compartment, which may boost the anti-tumor response.

Liver X receptors induce antiproliferative effects in basal-like breast cancer.

Liver X receptors (LXRs) are nuclear transcription factors important in the regulation of cholesterol transport, and glucose and fatty acid metabolism. The antiproliferative role of LXRs has been studied in a variety of malignancies and may represent a therapeutic opportunity in cancers lacking targeted therapies, such as triple-negative breast cancer. In this study, we investigated the impact of LXR agonists alone and in combination with carboplatin in preclinical models of breast cancer. In vitro experiments revealed a dose-dependent decrease in tumor cell proliferation in estrogen receptor-positive breast cancer cells, whereas LXR activation in vivo resulted in an increased growth inhibitory effect in a basal-like breast cancer model (in combination with carboplatin). Functional proteomic analysis identified differences in protein expression between responding and nonresponding models related to Akt activity, cell-cycle progression, and DNA repair. Furthermore, pathway analysis suggested that the LXR agonist in combination with carboplatin inhibits the activity of targets of E2F transcription factors and affects cholesterol homeostasis in basal-like breast cancer.

Biodistribution of Poly(alkyl cyanoacrylate) Nanoparticles in Mice and Effect on Tumor Infiltration of Macrophages into a Patient-Derived Breast Cancer Xenograft.

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects.

Mechanism of cellular uptake and cytotoxicity of paclitaxel loaded lipid nanocapsules in breast cancer cells.

Lipid nanocapsules (LNCs) have proven their efficacy in delivering different drugs to various cancers, but no studies have yet described their uptake mechanisms, paclitaxel (PTX) delivery or resulting cytotoxicity towards breast cancer cells. Herein, we report results concerning cellular uptake of LNCs and cytotoxicity studies of PTX-loaded LNCs (LNCs-PTX) on the three breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468. LNCs-PTX of sizes 50 ± 2 nm, 90 ± 3 nm and 120 ± 4 nm were developed by the phase inversion method. Fluorescence microscopy and flow cytometry were used to observe the uptake of fluorescently labeled LNCs and cellular uptake of LNCs-PTX was measured using HPLC analyses of cell samples. These studies revealed a higher uptake of LNCs-PTX in MDA-MB-468 cells than in the other two cell lines. Moreover, free PTX and LNCs-PTX exhibited a similar pattern of toxicity towards each cell line, but MDA-MB-468 cells appeared to be more sensitive than the other two cell lines, as evaluated by the MTT cytotoxicity assay and a cell proliferation assay based upon [3H]thymidine incorporation. Studies with inhibitors of endocytosis indicate that the cellular uptake is mainly via the Cdc42/GRAF-dependent endocytosis as well as by macropinocytosis, whereas dynamin-dependent processes are not required. Furthermore, our results indicate that endocytosis of LNCs-PTX is important for the toxic effect on cells. Western blot analysis revealed that LNCs-PTX induce cytotoxicity by means of apoptosis in all the three cell lines. Altogether, the results demonstrate that LNCs-PTX exploit different mechanisms of endocytosis in a cell-type dependent manner, and subsequently induce apoptotic cell death in the breast cancer cells here studied. The article also describes biodistribution studies following intravenous injection of fluorescently labeled LNCs in mice.

Physicochemical Characterization, Toxicity and In Vivo Biodistribution Studies of a Discoidal, Lipid-Based Drug Delivery Vehicle: Lipodisq Nanoparticles Containing Doxorubicin.

Many promising pharmaceutically active compounds have low solubility in aqueous environments and their encapsulation into efficient drug delivery vehicles is crucial to increase their bioavailability. Lipodisq nanoparticles are approximately 10 nm in diameter and consist of a circular phospholipid bilayer, stabilized by an annulus of SMA (a hydrolysed copolymer of styrene and maleic anhydride). SMA is used extensively in structural biology to extract and stabilize integral membrane proteins for biophysical studies. Here, we assess the potential of these nanoparticles as drug delivery vehicles, determining their cytotoxicity and the in vivo excretion pathways of their polymer and lipid components. Doxorubicin-loaded Lipodisqs were cytotoxic across a panel of cancer cell lines, whereas nanoparticles without the drug had no effect on cell proliferation. Intracellular doxorubicin release from Lipodisqs in HeLa cells occurred in the low-pH environment of the endolysosomal system, consistent with the breakdown of the discoidal structure as the carboxylate groups of the SMA polymer become protonated. Biodistribution studies in mice showed that, unlike other nanoparticles injected intravenously, most of the Lipodisq components were recovered in the colon, consistent with rapid uptake by hepatocytes and excretion into bile. These data suggest that Lipodisqs have the potential to act as delivery vehicles for drugs and contrast agents.

Paclitaxel-loaded biodegradable ROS-sensitive nanoparticles for cancer therapy.

BACKGROUND: Reactive oxygen species (ROS), such as hydrogen peroxide and superoxide, trigger biodegradation of polymer-based nanoparticles (NPs) bearing pinacol-type boronic ester groups. These NPs may selectively release their cargo, in this case paclitaxel (PTX), at the high levels of ROS present in the intracellular environment of inflamed tissues and most tumors. PURPOSE: The main objective was to determine anti-tumor efficacy of PTX-loaded ROS-sensitive NPs and to examine whether macrophage infiltration had any impact on treatment efficacy. METHODS: NPs were synthesized and their characteristics in the presence of H2O2 were demonstrated. Both confocal microscopy as well as flow cytometry approaches were used to determine degradation of ROS-sensitive NPs. HeLa cells were cultured in vitro and used to establish tumor xenografts in nude mice. In vivo experiments were performed to understand toxicity, biodistribution and anti-tumor efficacy of the NPs. Moreover, we performed immunohistochemistry on tumor sections to study infiltration of M1 and M2 subsets of macrophages. RESULTS: We demonstrated that PTX delivered in NPs containing a ROS-sensitive polymer exhibits a better anti-tumor efficacy than PTX in NPs containing ROS-non-sensitive polymer, free PTX or Abraxane® (nab-PTX). The biodistribution revealed that ROS-sensitive NPs exhibit retention in liver, spleen and lungs, suggesting a potential to target cancer metastasizing to these organs. Finally, we demonstrated a correlation between infiltrated macrophage subsets and treatment efficacy, possibly contributing to the efficient anti-tumor effects. CONCLUSION: Treatment with ROS-sensitive NPs containing PTX gave an improved therapeutic effect in HeLa xenografts than their counterpart, free PTX or nab-PTX. Our data revealed a correlation between macrophage infiltration and efficiency of the different antitumor treatments, as the most effective NPs resulted in the highest infiltration of the anti-tumorigenic M1 macrophages.

Cabazitaxel-loaded Poly(2-ethylbutyl cyanoacrylate) nanoparticles improve treatment efficacy in a patient derived breast cancer xenograft.

The effect of poly(2-ethyl-butyl cyanoacrylate) nanoparticles containing the cytotoxic drug cabazitaxel was studied in three breast cancer cell lines and one basal-like patient-derived xenograft model grown in the mammary fat pad of immunodeficient mice. Nanoparticle-encapsulated cabazitaxel had a much better efficacy than similar concentrations of free drug in the basal-like patient-derived xenograft and resulted in complete remission of 6 out of 8 tumors, whereas free drug gave complete remission only with 2 out of 9 tumors. To investigate the different efficacies obtained with nanoparticle-encapsulated versus free cabazitaxel, mass spectrometry quantification of cabazitaxel was performed in mice plasma and selected tissue samples. Nanoparticle-encapsulated drug had a longer circulation time in blood. There was approximately a three times higher drug concentration in tumor tissue 24 h after injection, and two times higher 96 h after injection of nanoparticles with drug compared to the free drug. The tissue biodistribution obtained after 24 h using mass spectrometry analyses correlates well with biodistribution data obtained using IVIS® Spectrum in vivo imaging of nanoparticles labeled with the fluorescent substance NR668, indicating that these data also are representative for the nanoparticle distribution. Furthermore, immunohistochemistry was used to estimate infiltration of macrophages into the tumor tissue following injection of nanoparticle-encapsulated and free cabazitaxel. The higher infiltration of anti-tumorigenic versus pro-tumorigenic macrophages in tumors treated with the nanoparticles might also contribute to the improved effect obtained with the nanoparticle-encapsulated drug. Tumor infiltration of pro-tumorigenic macrophages was four times lower when using nanoparticles containing cabazitaxel than when using particles without drug, and we speculate that the very good therapeutic efficacy obtained with our cabazitaxel-containing particles may be due to their ability to reduce the level of pro-tumorigenic macrophages in the tumor. In summary, encapsulation of cabazitaxel in poly(2-ethyl-butyl cyanoacrylate) nanoparticles seems promising for treatment of breast cancer.

Identification of human NK17/NK1 cells.

BACKGROUND: Natural killer (NK) cells have both cytolytic and immunoregulatory functions. We recently described that these cells release the inflammatory cytokines IL-17 and IFN-γ. However, the precise identity of the NK cell subset(s) that secrete these cytokines is not known. METHODOLOGY/PRINCIPAL FINDINGS: To isolate the cells secreting IL-17 and IFN-γ, we took advantage of the findings that Th17/Th1 cells express chemokine receptors. Therefore, CD56(+)NK cells were stained with antibodies against various chemokine receptors and intracellularly with antibodies toward IL-17 and IFN-γ. Consequently, we identified previously unrecognized subset of NK cells generated from normal human peripheral blood after activation with IL-2 but not PMA plus ionomycin. The cells are characterized by the expression of CD56(+) and CCR4(+), produce IL-17 and IFN-γ and are consequently named NK17/NK1 cells. They also express CD161, NKp30, NKp44, NKp46, NKG2D, CD158, CCL22, IL-2Rß and the common γ chain but not CD127 or IL-23R. Further, they possess T-bet and RORγt transcription factors. Antibodies to IL-1ß, IL-6, IL-21, or TGF-ß1 do not inhibit IL-2-induced generation of NK17/NK1 cells, suggesting that IL-2 has the capacity to polarize these cells. Notably, NK17/NK1 cells are abundant in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) without activation, and are generated from the peripheral blood of these patients after activation with IL-2. CONCLUSIONS/SIGNIFICANCE: NK17/NK1 cells identified here have not been previously described in healthy or MS patients.