Multiplexed bioluminescence imaging with a substrate unmixing platform.

Bioluminescent tools can illuminate cellular features in whole organisms. Multi-component tracking remains challenging, though, owing to a lack of well-resolved probes and long imaging times. To address the need for more rapid, quantitative, and multiplexed bioluminescent readouts, we developed an analysis pipeline featuring sequential substrate administration and serial image acquisition. Light output from each luciferin is layered on top of the previous image, with minimal delay between substrate delivery. A MATLAB algorithm was written to analyze bioluminescent images generated from the rapid imaging protocol and deconvolute (i.e., unmix) signals from luciferase-luciferin pairs. Mixtures comprising three to five luciferase reporters were readily distinguished in under 50 min; this same experiment would require days using conventional workflows. We further showed that the algorithm can be used to accurately quantify luciferase levels in heterogeneous mixtures. Based on its speed and versatility, the multiplexed imaging platform will expand the scope of bioluminescence technology.

Macromolecular assembly of bioluminescent protein nanoparticles for enhanced imaging.

Bioluminescence imaging has advantages over fluorescence imaging, such as minimal photobleaching and autofluorescence, and greater signal-to-noise ratios in many complex environments. Although significant achievements have been made in luciferase engineering for generating bright and stable reporters, the full capability of luciferases for nanoparticle tracking has not been comprehensively examined. In biocatalysis, enhanced enzyme performance after immobilization on nanoparticles has been reported. Thus, we hypothesized that by assembling luciferases onto a nanoparticle, the resulting complex could lead to substantially improved imaging properties. Using a modular bioconjugation strategy, we attached NanoLuc (NLuc) or Akaluc bioluminescent proteins to a protein nanoparticle platform (E2), yielding nanoparticles NLuc-E2 and Akaluc-E2, both with diameters of ∼45 â€‹nm. Although no significant differences were observed between different conditions involving Akaluc and Akaluc-E2, free NLuc at pH 5.0 showed significantly lower emission values than free NLuc at pH 7.4. Interestingly, NLuc immobilization on E2 nanoparticles (NLuc-E2) emitted increased luminescence at pH 7.4, and at pH 5.0 showed over two orders of magnitude (>200-fold) higher luminescence (than free NLuc), expanding the potential for imaging detection using the nanoparticle even upon endocytic uptake. After uptake by macrophages, the resulting luminescence with NLuc-E2 nanoparticles was up to 7-fold higher than with free NLuc at 48 â€‹h. Cells incubated with NLuc-E2 could also be imaged using live bioluminescence microscopy. Finally, biodistribution of nanoparticles into lymph nodes was detected through imaging using NLuc-E2, but not with conventionally-labeled fluorescent E2. Our data demonstrate that NLuc-bound nanoparticles have advantageous properties that can be utilized in applications ranging from single-cell imaging to in vivo biodistribution.

Transcriptome analysis of heterogeneity in mouse model of metastatic breast cancer.

BACKGROUND: Cancer metastasis is a complex process involving the spread of malignant cells from a primary tumor to distal organs. Understanding this cascade at a mechanistic level could provide critical new insights into the disease and potentially reveal new avenues for treatment. Transcriptome profiling of spontaneous cancer models is an attractive method to examine the dynamic changes accompanying tumor cell spread. However, such studies are complicated by the underlying heterogeneity of the cell types involved. The purpose of this study was to examine the transcriptomes of metastatic breast cancer cells using the well-established MMTV-PyMT mouse model. METHODS: Organ-derived metastatic cell lines were harvested from 10 female MMTV-PyMT mice. Cancer cells were isolated and sorted based on the expression of CD44low/EpCAMhigh or CD44high/EpCAMhigh surface markers. RNA from each cell line was extracted and sequenced using the NextSeq 500 Illumina platform. Tissue-specific genes were compared across the different metastatic and primary tumor samples. Reads were mapped to the mouse genome using STAR, and gene expression was quantified using RSEM. Single-cell RNA-seq (scRNA-seq) was performed on select samples using the ddSeq platform by BioRad and analyzed using Seurat v3.2.3. Monocle2 was used to infer pseudo-time progression. RESULTS: Comparison of RNA sequencing data across all cell populations produced distinct gene clusters. Differential gene expression patterns related to CD44 expression, organ tropism, and immunomodulatory signatures were observed. scRNA-seq identified expression profiles based on tissue-dependent niches and clonal heterogeneity. These cohorts of data were narrowed down to identify subsets of genes with high expression and known metastatic propensity. Dot plot analyses further revealed clusters expressing cancer stem cell and cancer dormancy markers. Changes in relevant genes were investigated across pseudo-time and tissue origin using Monocle2. These data revealed transcriptomes that may contribute to sub-clonal evolution and treatment evasion during cancer progression. CONCLUSIONS: We performed a comprehensive transcriptome analysis of tumor heterogeneity and organ tropism during breast cancer metastasis. These data add to our understanding of metastatic progression and highlight targets for breast cancer treatment. These markers could also be used to image the impact of tumor heterogeneity on metastases.

Rapid Multicomponent Bioluminescence Imaging via Substrate Unmixing.

Studies of biological function demand probes that can report on processes in real time and in physiological environments. Bioluminescent tools are uniquely suited for this purpose, as they enable sensitive imaging in cells and tissues. Bioluminescent reporters can also be monitored continuously over time without detriment, as excitation light is not required. Rather, light emission derives from luciferase-luciferin reactions. Several engineered luciferases and luciferins have expanded the scope of bioluminescence imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Conventional approaches image one luciferase at a time. Consequently, short-term changes in cell growth or gene expression cannot be easily captured. Here, we report a strategy for rapid, multiplexed imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent imaging in mice was also achieved on the minutes-to-hours time scale.

Preclinical and Dose-Finding Phase I Trial Results of Combined Treatment with a TORC1/2 Inhibitor (TAK-228) and Aurora A Kinase Inhibitor (Alisertib) in Solid Tumors.

PURPOSE: The purpose of this study was to evaluate the rational combination of TORC1/2 inhibitor TAK-228 and Aurora A kinase inhibitor alisertib in preclinical models of triple-negative breast cancer (TNBC) and to conduct a phase I dose escalation trial in patients with advanced solid tumors. EXPERIMENTAL DESIGN: TNBC cell lines and patient-derived xenograft (PDX) models were treated with alisertib, TAK-228, or the combination and evaluated for changes in proliferation, cell cycle, mTOR pathway modulation, and terminal cellular fate, including apoptosis and senescence. A phase I clinical trial was conducted in patients with advanced solid tumors treated with escalating doses of alisertib and TAK-228 using a 3+3 design to determine the maximum tolerated dose (MTD). RESULTS: The combination of TAK-228 and alisertib resulted in decreased proliferation and cell-cycle arrest in TNBC cell lines. Treatment of TNBC PDX models resulted in significant tumor growth inhibition and increased apoptosis with the combination. In the phase I dose escalation study, 18 patients with refractory solid tumors were enrolled. The MTD was alisertib 30 mg b.i.d. days 1 to 7 of a 21-day cycle and TAK-228 2 mg daily, continuous dosing. The most common treatment-related adverse events were neutropenia, fatigue, nausea, rash, mucositis, and alopecia. CONCLUSIONS: The addition of TAK-228 to alisertib potentiates the antitumor activity of alisertib in vivo, resulting in increased cell death and apoptosis. The combination is tolerable in patients with advanced solid tumors and should be evaluated further in expansion cohorts with additional pharmacodynamic assessment.

First-in-Class Phosphorylated-p68 Inhibitor RX-5902 Inhibits ß-Catenin Signaling and Demonstrates Antitumor Activity in Triple-Negative Breast Cancer.

RX-5902 is a first-in-class anticancer agent targeting phosphorylated-p68 and attenuating nuclear shuttling of ß-catenin. The purpose of this study was to evaluate the efficacy of RX-5902 in preclinical models of triple-negative breast cancer (TNBC) and to explore effects on ß-catenin expression. A panel of 18 TNBC cell lines was exposed to RX-5902, and changes in proliferation, apoptosis, cellular ploidy, and effector protein expression were assessed. Gene expression profiling was used in sensitive and resistant cell lines with pathway analysis to explore pathways associated with sensitivity to RX-5902. The activity of RX-5902 was confirmed in vivo in cell line and patient-derived tumor xenograft (PDX) models. RX-5902 demonstrated potent antiproliferative activity in vitro against TNBC cell lines with an average IC50 of 56 nmol/L in sensitive cell lines. RX-5902 treatment resulted in the induction of apoptosis, G2-M cell-cycle arrest, and aneuploidy in a subset of cell lines. RX-5902 was active in vivo against TNBC PDX models, and treatment resulted in a decrease in nuclear ß-catenin. RX-5902 exhibited dose-proportional pharmacokinetics and plasma and tumor tissue in nude mice. Pathway analysis demonstrated an increase in the epithelial-to-mesenchymal transformation (EMT), TGFß, and Wnt/ß-catenin pathways associated with sensitivity to RX-5902. RX-5902 is active against in vitro and in vivo preclinical models of TNBC. Target engagement was confirmed with decreases in nuclear ß-catenin and MCL-1 observed, confirming the proposed mechanism of action. This study supports the continued investigation of RX-5902 in TNBC and combinations with immunotherapy.

Efficacy and Molecular Mechanisms of Differentiated Response to the Aurora and Angiogenic Kinase Inhibitor ENMD-2076 in Preclinical Models of p53-Mutated Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is a subtype associated with poor prognosis and for which there are limited therapeutic options. The purpose of this study was to evaluate the efficacy of ENMD-2076 in p53-mutated TNBC patient-derived xenograft (PDX) models and describe patterns of terminal cell fate in models demonstrating sensitivity, intrinsic resistance, and acquired resistance to ENMD-2076. EXPERIMENTAL DESIGN: p53-mutated, TNBC PDX models were treated with ENMD-2076 and evaluated for mechanisms of sensitivity or resistance to treatment. Correlative tissue testing was performed on tumor tissue to assess for markers of proliferation, apoptosis, senescence, and pathways of resistance after treatment and at the time of acquired resistance. RESULTS: Sensitivity to ENMD-2076 200 mg/kg daily was associated with induction of apoptosis while models exhibiting intrinsic or acquired resistance to treatment presented with a senescent phenotype. Response to ENMD-2076 was accompanied by an increase in p53 and p73 levels, even within the background of mutant p53. Treatment with ENMD-2076 resulted in a decrease in pAurA and an increase in pHH3. We observed a TNBC subtype switch from the luminal androgen receptor to the basal-like subtype at acquired resistance. CONCLUSION: ENMD-2076 has antitumor activity in preclinical models of p53-mutated TNBC. Increased levels of p53 and p73 correlated with sensitivity whereas senescence was associated with resistance to ENMD-2076. The novel finding of a TNBC subtype switch at time of acquired resistance may provide mechanistic insights into the biologic effects of selective pressure of anticancer treatments on TNBC. ENMD-2076 is currently being evaluated in a Phase 2 clinical trial in patients with metastatic, previously treated TNBC where these biologic correlates can be further explored.

An integrated bioinformatics analysis to dissect kinase dependency in triple negative breast cancer.

BACKGROUND: Triple-Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis. Clinically, TNBC patients have limited treatment options besides chemotherapy. The goal of this study was to determine the kinase dependency in TNBC cell lines and to predict compounds that could inhibit these kinases using integrative bioinformatics analysis. RESULTS: We integrated publicly available gene expression data, high-throughput pharmacological profiling data, and quantitative in vitro kinase binding data to determine the kinase dependency in 12 TNBC cell lines. We employed Kinase Addiction Ranker (KAR), a novel bioinformatics approach, which integrated these data sources to dissect kinase dependency in TNBC cell lines. We then used the kinase dependency predicted by KAR for each TNBC cell line to query K-Map for compounds targeting these kinases. We validated our predictions using published and new experimental data. CONCLUSIONS: In summary, we implemented an integrative bioinformatics analysis that determines kinase dependency in TNBC. Our analysis revealed candidate kinases as potential targets in TNBC for further pharmacological and biological studies.

p53 Family Members Regulate Phenotypic Response to Aurora Kinase A Inhibition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. Advances in the treatment of TNBC have been hampered by the lack of novel effective targeted therapies. The primary goal of this study was to evaluate the efficacy of targeting Aurora kinase A (AurA), a key regulator of mitosis, in TNBC models. A secondary objective was to determine the role of the p53 family of transcriptional regulators, commonly mutated in TNBC, in determining the phenotypic response to the AurA inhibitor alisertib (MLN8237). Alisertib exhibited potent antiproliferative and proapoptotic activity in a subset of TNBC models. The induction of apoptosis in response to alisertib exposure was dependent on p53 and p73 activity. In the absence of functional p53 or p73, there was a shift in the phenotypic response following alisertib exposure from apoptosis to cellular senescence. In addition, senescence was observed in patient-derived tumor xenografts with acquired resistance to alisertib treatment. AurA inhibitors are a promising class of novel therapeutics in TNBC. The role of p53 and p73 in mediating the phenotypic response to antimitotic agents in TNBC may be harnessed to develop an effective biomarker selection strategy in this difficult to target disease.