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The epidermal growth factor receptor (EGFR) is a type I transmembrane protein, which is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. EGFR is a c...

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in a variety of cancer types, including breast, lung, gastric, pancreatic, and colorectal cancers. Trastuzumab, a...

Tepotinib is a highly selective and potent MET inhibitor in development for the treatment of patients with solid tumors. Given the favorable tolerability and safety profiles up to the maximum tested dose in the first‐in‐human (FIH) trial, an efficacy‐driven translational modeling approach was proposed to establish the recommended phase II dose (RP2D). To study the in vivo pharmacokinetics (PKs)/target inhibition/tumor growth inhibition relationship, a subcutaneous KP‐4 pancreatic cell‐line xenograft model in mice with sensitivity to MET pathway inhibition was selected as a surrogate tumor model. Further clinical PK and target inhibition data (derived from predose and postdose paired tumor biopsies) from a FIH study were integrated with the longitudinal PKs and target inhibition profiles from the mouse xenograft study to establish a translational PK/pharmacodynamic (PD) model. Preclinical data showed that tumor regression with tepotinib treatment in KP‐4 xenograft tumors corresponded to 95% target inhibition. We therefore concluded that a PD criterion of sustained, near‐to‐complete (>95%) phospho‐MET inhibition in tumors should be targeted for tepotinib to be effective. Simulations of dose‐dependent target inhibition profiles in human tumors that exceeded the PD threshold in more than 90% of patients established an RP2D of tepotinib 500 mg once daily. This translational mathematical modeling approach supports an efficacy‐driven rationale for tepotinib phase II dose selection of 500 mg once daily. Tepotinib at this dose has obtained regulatory approval for the treatment of patients with non‐small cell lung cancer harboring MET exon 14 skipping.

Background: Patients with osteoblastic bone metastases are candidates for radium-223 ((223)RaCl(2)) therapy and may undergo sodium fluoride-18 ((18)F-NaF) positron emission tomography-computed tomography imaging to identify bone lesions. (18)F-NaF has been shown to predict (223)RaCl(2) uptake, but intratumor distributions of these two agents remain unclear. In this study, the authors evaluate the spatial distribution and relative uptakes of (18)F-NaF and (223)RaCl(2) in Hu09-H3 human osteosarcoma mouse xenograft tumors at macroscopic and microscopic levels to better quantify their correlation. Materials and Methods: (18)F-NaF and (223)RaCl(2) were co-injected into Hu09-H3 xenograft tumor severe combined immunodeficient mice. Tumor content was determined from in vivo biodistributions and visualized by PET, single photon emission computed tomography, and CT imaging. Intratumor distributions were visualized by quantitative autoradiography of tumor tissue sections and compared to histology of the same or adjacent sections. Results: (18)F and (223)Ra accumulated in proportional amounts in whole Hu09-H3 tumors (r(2) = 0.82) and in microcalcified regions within these tumors (r(2) = 0.87). Intratumor distributions of (18)F and (223)Ra were spatially congruent in these microcalcified regions. Conclusions: (18)F-NaF and (223)RaCl(2) uptake are strongly correlated in heterogeneously distributed microcalcified regions of Hu09-H3 xenograft tumors, and thus, tumor accumulation of (18)F is predictive of (223)Ra accumulation. Hu09-H3 xenograft tumors appear to possess certain histopathological features found in patients with metastatic bone disease and may be useful in clarifying the relationship between administered (223)Ra dose and therapeutic effect.

Providing physicians with new imaging agents to help detect cancer with better sensitivity and specificity has the potential to significantly improve patient outcomes. Development of new imaging agents could offer improved early cancer detection during routine screening or help surgeons identify tumor margins for surgical resection. In this study, we evaluate the optical properties of a colorful class of dyes and pigments that humans routinely encounter. The pigments are often used in tattoo inks and the dyes are FDA approved for the coloring of foods, drugs, and cosmetics. We characterized their absorption, fluorescence and Raman scattering properties in the hopes of identifying a new panel of dyes that offer exceptional imaging contrast. We found that some of these coloring agents, coined as "optical inks", exhibit a multitude of useful optical properties, outperforming some of the clinically approved imaging dyes on the market. The best performing optical inks (Green 8 and Orange 16) were further incorporated into liposomal nanoparticles to assess their tumor targeting and optical imaging potential. Mouse xenograft models of colorectal, cervical and lymphoma tumors were used to evaluate the newly developed nano-based imaging contrast agents. After intravenous injection, fluorescence imaging revealed significant localization of the new "optical ink" liposomal nanoparticles in all three tumor models as opposed to their neighboring healthy tissues (p < 0.05). If further developed, these coloring agents could play important roles in the clinical setting. A more sensitive imaging contrast agent could enable earlier cancer detection or help guide surgical resection of tumors, both of which have been shown to significantly improve patient survival.

P-cadherin-LP-DART is a bispecific antibody targeting P-cadherin expressed on the tumor cells and CD3 on the T-cells. Previously we demonstrated the development and efficacy of P-cadherin-LP-DART in in vitro and in vivo models. Here, we evaluated the three pillars: exposure, targeting specificity and pharmacodynamic modulation for P-cadherin-LP-DART using fluorescence molecular tomography (FMT). Bispecific antibodies and T-cells were conjugated with a near-infrared fluorophores: VivoTag®680XL (VT680) and CellVue®NIR815 (CV815), respectively. In vitro binding and cytotoxic T-lymphocyte assay demonstrated that P-cadherin-LP-DART significantly retained its properties after VT680 conjugation. In vivo FMT imaging was performed to determine the bispecific biodistribution and T-cell trafficking in HCT-116 xenograft model. Peak tumor exposure (2.71%ID) was observed at 96 hr post-injection with measurable quantity even at 240 hr (1.46%ID) (Pillar 1). P-cadherin-LP-DART accumulation in tumor was 20-25 fold higher compared to Control-LP-DART demonstrating the targeting specificity (Pillar 2). Imaging after engraftment of CV815 labeled T-cells showed P-cadherin-LP-DART mediated T-cell trafficking in tumors (Pillar 3). This study harnessed the multichannel capability of FMT and demonstrated the targeting of drug and trafficking of T cells to tumors, simultaneously. Our results show the impact of molecular imaging in demonstrating three pillars of pharmacology, longitudinally and non-invasively.

Background: Despite active studies of the clinical importance of BRAFV600E, suitable research models to investigate the role of this mutation in the etiopathogenesis of human thyroid cancers are li...

Hepatocellular carcinoma (HCC) is responsible for the second-leading cancer-related death toll worldwide. Although sorafenib and levantinib as frontline therapy and regorafenib, cabazantinib and ramicurimab have now been approved for second-line therapy, the therapeutic benefit is in the range of only a few months with respect to prolongation of survival. Aggressiveness of HCC is mediated by metastasis. Intrahepatic metastases and distant metastasis to the lungs, lymph nodes, bones, omentum, adrenal gland and brain have been observed. Therefore, the identification of metastasis-related new targets and treatment modalities is of paramount importance. In this review, we focus on metastasis-related microRNAs (miRs) as therapeutic targets for HCC. We describe miRs which mediate or repress HCC metastasis in mouse xenograft models. We discuss 18 metastasis-promoting miRs and 35 metastasis-inhibiting miRs according to the criteria as outlined. Six of the metastasis-promoting miRs (miR-29a, -219-5p, -331-3p, 425-5p, -487a and -1247-3p) are associated with unfavourable clinical prognosis. Another set of six down-regulated miRs (miR-101, -129-3p, -137, -149, -503, and -630) correlate with a worse clinical prognosis. We discuss the corresponding metastasis-related targets as well as their potential as therapeutic modalities for treatment of HCC-related metastasis. A subset of up-regulated miRs -29a, -219-5p and -425-5p and down-regulated miRs -129-3p and -630 were evaluated in orthotopic metastasis-related models which are suitable to mimic HCC-related metastasis. Those miRNAs may represent prioritized targets emerging from our survey.

Arginine vasopressin (AVP) and its type-2 receptor (V2R) play an essential role in the regulation of salt and water homeostasis by the kidneys. V2R activation also stimulates proliferation of renal cell carcinoma (RCC) cell lines in vitro. The current studies investigated V2R expression and activity in human RCC tumors, and its role in RCC tumor growth. Examination of the cancer genome atlas (TCGA) database, and analysis of human RCC tumor tissue microarrays, cDNA arrays and tumor biopsy samples demonstrated V2R expression and activity in clear cell RCC (ccRCC). In vitro, V2R antagonists OPC31260 and Tolvaptan, or V2R gene silencing reduced wound closure and cell viability of 786-O and Caki-1 human ccRCC cell lines. Similarly in mouse xenograft models, Tolvaptan and OPC31260 decreased RCC tumor growth by reducing cell proliferation and angiogenesis, while increasing apoptosis. In contrast, the V2R agonist dDAVP significantly increased tumor growth. High intracellular cAMP levels and ERK1/2 activation were observed in human ccRCC tumors. In mouse tumors and Caki-1 cells, V2R agonists reduced cAMP and ERK1/2 activation, while dDAVP treatment had the reverse effect. V2R gene silencing in Caki-1 cells also reduced cAMP and ERK1/2 activation. These results provide novel evidence for a pathogenic role of V2R signaling in ccRCC, and suggest that inhibitors of the AVP-V2R pathway, including the FDA-approved drug Tolvaptan, could be utilized as novel ccRCC therapeutics.

Glyceollins are soybean-derived phytoalexins that induce the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway, which is involved in the detoxification of carcinogens an...

Gemcitabine (GEM) is a first-line drug for pancreatic cancer therapy, but GEM resistance is easily developed in patients. Growing evidence suggests that cancer chemoprevention and suppression are highly associated with dietary phytochemical and microbiota composition. Ursolic acid (UA) has anti-inflammatory and anticancer effects; however, its role in improving cancer drug resistance in vivo remains unclear. In this study, the aim was to explore the role of UA in managing drug resistance-associated molecular mechanisms and the influence of gut microbiota. The in vitro results showed that receptor for advanced glycation end products (RAGE), nuclear factor kappa B p65 (NF-κB/p65), and multidrug resistance protein 1 (MDR1) protein levels were significantly increased in GEM-resistant pancreatic cancer cells (named MIA PaCa-2

Algae are the basis of primary production in aquatic ecosystems. The onset of food production in a aquatic environment begins with these organisms and with a complex mechanism called photosynthesis...

Aims: This work aimed to establish an accelerated imaging system for redox-sensitive mapping in a mouse tumor model using electron paramagnetic resonance (EPR) and nitroxyl radicals. Results: Sparse sampling of EPR spectral projections was demonstrated for a solution phantom. The reconstructed three-dimensional (3D) images with filtered back-projection (FBP) and compressed sensing image reconstruction were quantitatively assessed for the solution phantom. Mouse xenograft models of a human-derived pancreatic ductal adenocarcinoma cell line, MIA PaCa-2, were also measured for redox-sensitive mapping with the sparse sampling technique. Innovation: A short-lifetime redox-sensitive nitroxyl radical (N-15-labeled perdeuterated Tempone) could be measured to map the decay rates of the EPR signals for the mouse xenograft models. Acceleration of 3D EPR image acquisition broadened the choices of nitroxyl radical probes with various redox sensitivities to biological environments. Conclusion: Sparse sampling of EPR spectral projections accelerated image acquisition in the 3D redox-sensitive mapping of mouse tumor-bearing legs fourfold compared with conventional image acquisition with FBP.

BACKGROUND Dutasteride has been shown to increase expression of the prostate-specific membrane antigen (PSMA) in prostate cancer cells in previous in vitro studies. This 5-alpha-reductase inhibitor is commonly used for the treatment of symptomatic benign prostatic enlargement. The modulation of PSMA expression might affect PSMA-based prostate cancer imaging and therapy. AIM The purpose of this work was to further analyze concentration-dependent effects of Dutasteride on PSMA expression in a mouse xenograft model. METHODS AND RESULTS Four groups of mice bearing LNCaP xenografts were treated for 14 days with daily intraperitoneal injections of either vehicle control or different concentrations of Dutasteride (0.1, 1, 10 mg/kg). Total expression of PSMA, androgen receptor (AR), and caspase-3 protein was analyzed using immunoblotting (WES). In addition, PSMA, cleaved caspase-3 and Ki-67 expression was assessed and quantified by immunohistochemistry. Tumor size was measured by caliper on day 7 and 14, tumor weight was assessed following tissue harvesting. The mean PSMA protein expression in mice increased significantly after treatment with 1 mg/kg (10-fold) or 10 mg/kg (sixfold) of Dutasteride compared to vehicle control. The mean fluorescence intensity significantly increased by daily injections of 0.1 mg/kg Dutasteride (1.6-fold) as well as 1 and 10 mg/kg Dutasteride (twofold). While the reduction in tumor volume following treatment with high concentrations of 10 mg/kg Dutasteride was nonsignificant, no changes in AR, caspase-3, cleaved caspase-3, and Ki-67 expression were observed. CONCLUSION Short-term Dutasteride treatments with concentrations of 1 and 10 mg/kg significantly increase the total PSMA protein expression in a mouse LNCaP xenograft model. PSMA fluorescence intensity increases significantly even using lower daily concentrations of 0.1 mg/kg Dutasteride. Further investigations are needed to elucidate the impact of Dutasteride treatment on PSMA expression in patients.

Heterobifunctional compounds that direct the ubiquitination of intracellular proteins in a targeted manner via co-opted ubiquitin ligases have enormous potential to transform the field of medicinal chemistry. These chimeric molecules, often termed proteolysis-targeting chimeras (PROTACs) in the chemical literature, enable the controlled degradation of specific proteins via their direction to the cellular proteasome. In this report, we describe the second phase of our research focused on exploring antibody-drug conjugates (ADCs), which incorporate BRD4-targeting chimeric degrader entities. We employ a new BRD4-binding fragment in the construction of the chimeric ADC payloads that is significantly more potent than the corresponding entity utilized in our initial studies. The resulting BRD4-degrader antibody conjugates exhibit potent and antigen-dependent BRD4 degradation and antiproliferation activities in cell-based experiments. Multiple ADCs bearing chimeric BRD4-degrader payloads also exhibit strong, antigen-dependent antitumor efficacy in mouse xenograft assessments that employ several different tumor models.

Simple Summary Studying rare outcomes in cancer is challenging because observation of the rare event may require a very high number of patients or experimental animals. Here, we propose a new, predictive approach to understanding the biological mechanisms underlying such rare events in cancer treatment outcomes. We take as a case-study, the treatment of metastatic, castration-resistant prostate cancer with the live cell vaccine, sipuleucel-t (Provenge). Only a fraction of patients benefited from Provenge; that is, clinical success is a rare event. It remains an open question why Provenge conferred such a modest survival benefit. Our modeling paradigm captures the inherent heterogeneity that characterizes individuals in a population, and provides an explanation for the observed clinical outcomes of treatment with Provenge. Our approach readily generalizes to a range of emerging cancer immunotherapies, and more generally, to predicting and understanding how a population responds to any intervention targeting a human disease. Abstract Sipuleucel-T (Provenge) is the first live cell vaccine approved for advanced, hormonally refractive prostate cancer. However, survival benefit is modest and the optimal combination or schedule of sipuleucel-T with androgen depletion remains unknown. We employ a nonlinear dynamical systems approach to modeling the response of hormonally refractive prostate cancer to sipuleucel-T. Our mechanistic model incorporates the immune response to the cancer elicited by vaccination, and the effect of androgen depletion therapy. Because only a fraction of patients benefit from sipuleucel-T treatment, inter-individual heterogeneity is clearly crucial. Therefore, we introduce our novel approach, Standing Variations Modeling, which exploits inestimability of model parameters to capture heterogeneity in a deterministic model. We use data from mouse xenograft experiments to infer distributions on parameters critical to tumor growth and to the resultant immune response. Sampling model parameters from these distributions allows us to represent heterogeneity, both at the level of the tumor cells and the individual (mouse) being treated. Our model simulations explain the limited success of sipuleucel-T observed in practice, and predict an optimal combination regime that maximizes predicted efficacy. This approach will generalize to a range of emerging cancer immunotherapies.

Manganese dioxide (MnO2) nanoparticles (NPs) are highly attractive for biomedical applications due to their biocompatibility, stimuli-responsive magnetic resonance imaging (MRI) properties and capability to modulate the hypoxic tumour microenvironment (TME). However, conventional MnO2 NPs do not possess photothermal therapy (PTT) functions except for hybrids with other photothermal materials. Herein, we first reveal the extraordinary photothermal conversion efficiency (44%) of enzymatically synthesised MnO2 NPs (Bio-MnO2 NPs), which are distinct from chemically synthesised MnO2 NPs. In addition, the Bio-MnO2 NPs revealed high thermal recycling stability and solubility as well as dual pH- and reduction-responsive MRI enhancement for tumour theragnosis. These NPs were prepared through a facile MnxEFG enzyme-mediated biomineralization process. The MnxEFG complex from Bacillus sp. PL-12 is the only manganese mineralization enzyme that could be heterologously overexpressed in its active form to achieve Bio-MnO2 NPs without a bacterial host. The hexagonal layer symmetry of the Bio-MnO2 NPs is the key feature facilitating the high photothermal conversion efficiency and TME-responsive T1-weighted MRI. Evaluations both in vitro at the cellular level and in vivo in a systematic tumour-bearing mouse xenograft model demonstrated the high photothermal ablation efficacy of the Bio-MnO2 NPs, which achieved complete tumour eradication with high therapeutic biosafety without obvious reoccurrence. Moreover, stimuli-responsive MR enhancement potentially allows imaging-guided precision PTT. With their excellent biocompatibility, mild synthesis conditions and relatively simple composition, Bio-MnO2 NPs hold great translational promise.

Proteases can play essential roles in severe human pathology, ranging from degenerative and inflammatory illnesses to infectious diseases, with some, such as Taspase1, involved in growth and progression of tumors at primary and metastatic sites. Taspase1 is a N-terminal nucleophile (Ntn)-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cellsin vitroand in mouse xenograft models of glioblastoma, thus this protein has the potential to become a novel anticancer drug target. It belongs to the family of Ntn-hydrolases, a unique family of proteins synthesized as enzymatically inactive proenzymes that become activated upon cleavage of the peptide bond on the N-terminal side of a threonine residue, which then becomes the catalytic site nucleophile. The activation process simultaneously changes the conformation of a long domain at the C-terminus of the alpha-subunit for which no full-length structural information exists and its function is poorly understood. Here we present a novel cloning strategy to generate a fully active, circularly permuted form of Taspase1 to determine the crystallographic structure of catalytically active human Taspase1 to 3.04Å. We discovered that this region forms a long helical domain and is indispensable for the catalytic activity of Taspase1. Together, our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases and suggests that this long domain could be a novel target for the design of inhibitors with the potential to be developed into anticancer therapeutics.

We developed a novel dividing device that can split needle biopsy tissues along longitude axis aiming to achieve definitive molecular-biological and genetical analysis with reference of pathological diagnosis of the side-by-side divided tissue as spatially matched information. The aim of this study was to evaluate the feasibility and potential usefulness of the novel dividing device to provide the appropriate materials for molecular diagnosis. The new device was examined using mouse xenograft tumors. Real-time quantitative PCR and genetic test were performed to evaluate the feasibility and usefulness of the device. All the samples from needle biopsy were successfully divided into two pieces. Quality and quantity from divided samples harbor high enough to perform gene expression analysis (real-time PCR) and genetic test. Using two divided samples obtained from xenograft tumor model by needle biopsy, the % length of xenograft tumor (human origin) was significantly correlated with the % human genomic DNA (p = 0.00000608, r = 0.987), indicating that these divided samples were spatially matched. The novel longitudinally dividing device of a needle biopsy tissue was useful to provide the appropriate materials for molecular-biological and genetical analysis with reference of pathological diagnosis as spatially matched information.

Numerical chromosome instability, or nCIN, defined as the high frequency of whole chromosome gains and losses, is prevalent in many solid tumors. nCIN has been shown to promote intra-tumor heterogeneity and corresponds with tumor aggressiveness, drug resistance and tumor relapse. While increased nCIN has been shown to promote the acquisition of genomic changes responsible for drug resistance, the potential to modulate nCIN in a therapeutic manner has not been well explored. Here we assess the role of nCIN in the acquisition of drug resistance in non small cell lung cancer. We show that generation of whole chromosome segregation errors in non small cell lung cancer cells is sensitive to manipulation of microtubule dynamics and that enhancement of chromosome cohesion strongly suppresses nCIN and reduces intra-tumor heterogeneity. We demonstrate that suppression of nCIN has no impact on non small cell lung cancer cell proliferation in vitro nor in tumor initiation in mouse xenograft models. However, suppression of nCIN alters the timing and molecular mechanisms that drive acquired drug resistance. These findings suggest mechanisms to suppress nCIN may serve as effective co-therapies to limit tumor evolution and sustain drug response.Statement of SignificanceModulation of microtubule dynamics in cells that exhibit chromosome instability (CIN) is sufficient to promote mitotic fidelity, reduce genomic heterogeneity, and limit acquisition of drug resistance.

Glioma-associated oncogene family zinc finger 2 (Gli2), a key component of the hedgehog signaling pathway, has been previously demonstrated to promote the malignant properties of prostate cancer in vitro. However, the role of Gli2 in the development of castration-resistant prostate cancer (CRPC) has yet to be fully elucidated. In the present study, Gli2 expression was knocked down in androgen-responsive prostate cancer cells using an inducible Gli2 short hairpin RNA. Suppression of Gli2 expression resulted in significant reduction of cell viability, increased the proportion of cells in the G0/G1 phases of the cell cycle and reduced the expression of genes associated with cell cycle progression. Gli2 knockdown sensitized both androgen-dependent and -independent prostate cancer cells to the antiandrogen drug Casodex and prevented the outgrowth of LNCaP prostate cancer cells. In addition, Gli2 knockdown significantly suppressed the development of CRPC in a LNCaP xenograft mouse model, which was reversed by the re-expression of Gli2. In conclusion, to the best of our knowledge, the present study was the first occasion in which the essential role of Gli2 in the development of CRPC was demonstrated, providing a potential therapeutic target for the intervention of CRPC.

Multiplexed asymmetric-detection time-stretch optical microscopy (multi-ATOM) has recently been developed to enable high-throughput quantitative phase imaging flow cytometry, from which single-cell biophysical properties can be measured at large scale. However, it lacks the ability to link such biophysical knowledge to biomolecular signatures at the single-cell precision for validation and correlative multi-scale single-cell analysis. We report a high-throughput multimodal system that integrates multi-ATOM with multiplexed 1-D fluorescence imaging/detection, termed FluorATOM; and applied it to perform synchronized biophysical and biomolecular phenotyping of rare breast circulating tumor cells detected in peripheral blood in a mouse xenograft at a throughput of >10,000 cell/sec.

Near-infrared (NIR) activatable upconversion nanoparticles (UCNPs) enable wireless-based phototherapies by converting deep-tissue-penetrating NIR to visible light. UCNPs are therefore ideal as wireless transducers for photodynamic therapy (PDT) of deep-sited tumors. However, the retention of unsequestered UCNPs in tissue with minimal options for removal limits their clinical translation. To address this shortcoming, biocompatible UCNPs implants are developed to deliver upconversion photonic properties in a flexible, optical guide design. To enhance its translatability, the UCNPs implant is constructed with an FDA-approved poly(ethylene glycol) diacrylate (PEGDA) core clad with fluorinated ethylene propylene (FEP). The emission spectrum of the UCNPs implant can be tuned to overlap with the absorption spectra of the clinically relevant photosensitizer, 5-aminolevulinic acid (5-ALA). The UCNPs implant can wirelessly transmit upconverted visible light till 8 cm in length and in a bendable manner even when implanted underneath the skin or scalp. With this system, it is demonstrated that NIR-based chronic PDT is achievable in an untethered and noninvasive manner in a mouse xenograft glioblastoma multiforme (GBM) model. It is postulated that such encapsulated UCNPs implants represent a translational shift for wireless deep-tissue phototherapy by enabling sequestration of UCNPs without compromising wireless deep-tissue light delivery. Ministry of Education (MOE) Nanyang Technological University This work was supported by National University of Singapore’s start up grants, R-183-000-413-733 and R-185-000-363-733; Singapore’s Ministry of Education grants (MOE 2016-T3-1-004, R-397-000-274-112, R-397-000-270-114); and a Lee Kong Chian School of Medicine, Nanyang Technological University start-up grant.

A far-red light–activated split-Cas9 (FAST) system was developed for controlling gene editing in both mammalian cells and mice., It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)–activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode–based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.

The analysis of heterogeneous subpopulations of circulating tumor cells (CTCs) is critical to enhance our understanding of cancer metastasis and enable non-invasive cancer diagnosis and monitoring. The phenotypic variability and plasticity of these cells – properties closely linked to their clinical behavior – demand techniques that isolate viable, discrete fractions of tumor cells for functional assays of their behavior and detailed analysis of biochemical properties. Here, we introduce the Prism Chip, a high-resolution immunomagnetic profiling and separation chip which harnesses a cobalt-based alloy to separate a flowing stream of nanoparticle-bound tumor cells with differential magnetic loading into ten discrete streams. Using this approach, we achieve exceptional purity (5.7 log white blood cell depletion) of isolated cells. We test the differential profiling function of the integrated device using prostate cancer blood samples from a mouse xenograft model. Using integrated graphene Hall sensors, we demonstrate concurrent automated profiling of single cells and CTC clusters that belong to distinct subpopulations based on protein surface expression.

Despite the role of STAT3 as a known driver of oncogenesis, efforts to develop therapeutic STAT3 inhibitors have thus far been unsuccessful. In this issue of Cancer Cell, Bai et al. report a potent and selective STAT3 degrader capable of producing complete and long-lasting tumor regression in mouse xenograft models.

// Shanchun Guo 1, 2 , Changde Zhang 1, 2 , Melyssa Bratton 2, 3 , Madhusoodanan Mottamal 1, 2 , Jiawang Liu 1, 2 , Peng Ma 2, 3 , Shilong Zheng 1, 2 , Qiu Zhong 1, 2 , Lin Yang 4 , Thomas E. Wiese 2, 3 , Yong Wu 5 , Matthew J. Ellis 6 , Margarite Matossian 7 , Matthew E. Burow 7 , Lucio Miele 8 , Rene Houtman 9 and Guangdi Wang 1, 2 1 Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA 2 RCMI Cancer Research Center, Xavier University of Louisiana, New Orleans, LA 70125, USA 3 College of Pharmacy, Xavier University of Louisiana, New Orleans, LA 70125, USA 4 College of Pharmacy Chongqing Medical and Pharmaceutical College, University Town, Chongqing, 401331, China 5 Department of Internal Medicine, Charles Drew University, Los Angeles, CA 90059, USA 6 Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA 7 Section of Hematology & Medical Oncology, Tulane University School of Medicine, New Orleans, LA 70112, USA 8 Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA 9 Nuclear Receptor Group, PamGene International, 5211HH Den Bosch, The Netherlands Correspondence to: Guangdi Wang, email: gwang@xula.edu Keywords: steroidal oral SERD; breast cancer; estrogen receptor mutant; Y537S; bioavailability Received: October 12, 2017 Accepted: December 27, 2017 Published: January 08, 2018 ABSTRACT Advances in oral SERDs development so far have been confined to nonsteroidal molecules such as those containing a cinnamic acid moiety, which are in earlystage clinical evaluation. ZB716 was previously reported as an orally bioavailable SERD structurally analogous to fulvestrant. In this study, we examined the binding details of ZB716 to the estrogen receptor alpha (ERα) by computer modeling to reveal its interactions with the ligand binding domain as a steroidal molecule. We also found that ZB716 modulates ERα-coregulator interactions in nearly identical manner to fulvestrant. The ability of ZB716 to inhibit cell growth and downregulate ER expression in endocrine resistant, ERα mutant breast cancer cells was demonstrated. Moreover, in both the MCF-7 xenograft and a patient derived xenograft model, orally administered ZB716 showed superior efficacy in blocking tumor growth when compared to fulvestrant. Importantly, such enhanced efficacy of ZB716 was shown to be attributable to its markedly higher bioavailability, as evidenced in the final plasma and tumor tissue concentrations of ZB716 in mice where drug concentrations were found significantly higher than in the fulvestrant treatment group.

Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software "Cancer Cell Finder" that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.

Introduction Quantitative small animal radionuclide imaging studies are often carried out with the intention of estimating the total radioactivity content of various tissues such as the radioactivity content of mouse xenograft tumors exposed to putative diagnostic or therapeutic agents. We show that for at least one specific application, positron projection imaging (PPI) and PET yield comparable estimates of absolute total tumor activity and that both of these estimates are highly correlated with direct well-counting of these same tumors. These findings further suggest that in this particular application, PPI is a far more efficient data acquisition and processing methodology than PET. Methods Forty-one athymic mice were implanted with PC3 human prostate cancer cells transfected with prostate-specific membrane antigen (PSMA (+)) and one additional animal (for a total of 42) with a control blank vector (PSMA (−)). All animals were injected with [ 18 F] DCFPyl, a ligand for PSMA, and imaged for total tumor radioactivity with PET and PPI. The tumors were then removed, assayed by well counting for total radioactivity and the values between these methods intercompared. Results PET, PPI and well-counter estimates of total tumor radioactivity were highly correlated (R 2 > 0.98) with regression line slopes near unity (0.95 Conclusion Total mouse xenograft tumor radioactivity can be measured with PET or PPI with an accuracy comparable to well counting if certain experimental and pharmacokinetic conditions are met. In this particular application, PPI is significantly more efficient than PET in making these measurements.

A protein structure-guided drug design approach was employed to develop small molecule inhibitors of the BET family of bromodomains that were distinct from the known (+)-JQ1 scaffold class. These efforts led to the identification of a series of substituted benzopiperazines with structural features that enable interactions with many of the affinity-driving regions of the bromodomain binding site. Lipophilic efficiency was a guiding principle in improving binding affinity alongside drug-like physicochemical properties that are commensurate with oral bioavailability. Derived from this series was tool compound FT001, which displayed potent biochemical and cellular activity, translating to excellent in vivo activity in a mouse xenograft model (MV-4-11).

Herein we disclose SAR studies of a series of dimethylamino pyrrolidines which we recently reported as novel inhibitors of the PRC2 complex through disruption of EED/H3K27me3 binding. Modification of the indole and benzyl moieties of screening hit 1 provided analogs with substantially improved binding and cellular activities. This work culminated in the identification of compound 2, our nanomolar proof-of-concept (PoC) inhibitor which provided on-target tumor growth inhibition in a mouse xenograft model. X-ray crystal structures of several inhibitors bound in the EED active-site are also discussed.

Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Unfortunately reliable and reproducible methods for testing drugs against metastasis are not available. We have previously developed a robust metastatic model in which mice are pretreated with tumor cell-conditioned media (TCM) from human TNBC cells (MDA-MB-231 and SUM149) for 2 weeks prior to tumor cell inoculation. In this model we found reproducible metastases in lymph nodes (LN) and lungs within 4-5 weeks after orthotopic tumor inoculation [1]. We have discovered that the TNBC cells secrete large amounts of interleukin-6 (IL-6) that “educates” lymphatic endothelial cells (LEC) in the LN and lungs. Stat3, a transcription factor, gets activated and induces the synthesis of CCL5 and VEGF among other factors. CCL5 recruits the tumor cells to the LN and lungs; VEGF helps build blood vessels in the LN to facilitate tumor cell survival; VEGF produced in the lung helps the tumor cells extravasate into the lung. We have confirmed the importance of these factors by showing that inhibitors of these factors significantly inhibit metastasis. In this report, using Maraviroc (CCR5 inhibitor) and cMR16-1 Ab (murine surrogate of the anti-IL-6R antibody), we investigated the effect of the combination treatment on the tumor growth and metastasis of orthotopic tumor xenografts generated from MDA-MB-231-Luc-D3H2LN cells. 2x106 TNBC cells tagged with luciferase were suspended in 100 μl PBS/Matrigel (1:1) and injected s.c. into female 3- to 4-week-old BALB/c nu/nu athymic mice pretreated with tumor cell-conditioned media (TCM) from TNBC cells for 2 weeks. We administered the Maraviroc (8 mg/kg Maraviroc, orally daily) and cMR16-1 Ab (i.p. 3-days per week) for 5 weeks. Our data show that tumor growth was dramatically inhibited by cMR16-1 Ab. Further, the drug combination of Maraviroc with cMR16-1 Ab caused significant reduction of TNBC tumor growth in mice compared to single agents. In addition, we measured thoracic metastases by adding luciferin to mice and measuring luminescence ex-vivo in the IVIS imager. Significantly, both single treatment of Maraviroc and the combination of Maraviroc with cMR16-1 abrogated the thoracic metastasis compared to control and single treatment of cMR16-1. These findings implicate IL-6 and CCL5 signaling as a critical event in TNBC tumor growth and metastasis via crosstalk between cancer cells and stromal components. Further, these studies suggest that IL-6 and CCL5 act as key regulators orchestrating TNBC metastatic breast cancer. Therefore, we have provided evidence that supports the hypothesis that functional inhibition of the IL-6 and CCL-5 signaling pathway has the potential to circumvent TNBC growth and metastasis. [1] E Lee et al. Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis. Nat Commun. 2014 5:4715. Citation Format: Jin K, Popel AS. Synergistic effect of combinatorial treatment with maraviroc and tocilizumab on TNBC tumor growth and metastasis in mouse xenograft model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-09.

Background: Hepatocellular carcinoma (HCC) is the most common liver cancer and the second most lethal tumor worldwide. Although the treatment strategies are improved constantly, overall 5-years’ survival rates still remain devastating. One of the major obstacles to improve the prognosis of HCC patient is the extraordinary tumor heterogeneity. The efficacy of empirical treatment using targeted drugs for advanced patients varies significantly among HCC individuals due to their heterogenous nature. Currently, models of patient-derived xenografts (PDXs) and in vitro cancer organoids have potentials for patient-specific drug screening aimed at personalized medicine. However, they are still required significant improvements before practical applications. On the other hand, endeavors for study on new model system are also in pursuit for reaching a systematic revolutionary improvement in the field of studying HCC. Methods: Here, We adopted an engineered three-dimensional (3D) bio-printing technique for the first time in the field of HCC studying, and successfully established 3D bio-printed HCC (3DP-HCC) models from specimens of six HCC patients. Findings: These 3DP-HCC models grew well during long-term culture and retained the features of HCC, including stable expression of the specific biomarker for HCC as well as the stable maintenances of both genetic alterations and expression profiles of the original tumors. In the tests of empirical drugs screenings, our 3DP-HCC models convincingly confirmed the efficacy of specific candidate drugs for particular individuals of HCC patients. In addition, xenograft study also confirmed both tumorigenic potential and histological features were well retained by 3DP-HCC models after long-term culture. Interpretation: 3DP-HCC models are reliable in culture and helpful to predict patient-specific drugs for personalized medicine. Funding Statement: This work was supported by grants from the CAMS Innovation Fund for Medical Sciences (CIFMS) (No.2016-I2M-1-001) and Tsinghua University-Peking Union Medical College Hospital Cooperation Project (PTQH201904552). This work was also supported by grants from the Ministry of Science and Technology of China (MOST; 2019YFA0801501, 2016YFA0100500), the National Natural Science Foundation of China (NSFC; 31970687, 31571509, 31522038, 51805294, 81730078), CAMS Clinical and Translational Medicine Research Funds (2019XK320006), the projects of Fundamental Research of Shenzhen (JCYJ20170412101508433, JCYJ20180507183655307) and International Science and Technology Cooperation Projects (2016YFE0107100). Declaration of Interests: All of the authors have no conflicts of interest to disclose. Ethics Approval Statement: Mouse xenograft experiment was approved by the Peking Union Medical Hospital Animal Welfare and Ethical Board and was performed in accordance with the institutional regulation.

Motivation Circulating tumor cells (CTCs) are widely studied using liquid biopsy methods that analyze fractionally-small peripheral blood (PB) samples. However, little is known about natural fluctuations in CTC numbers that may occur over short timescales in vivo, and how these may affect detection and enumeration of rare CTCs from small blood samples. Methods We recently developed an optical instrument called "diffuse in vivo flow cytometry" (DiFC) that uniquely allows continuous, non-invasive counting of rare, green fluorescent protein expressing CTCs in large blood vessels in mice. Here, we used DiFC to study short-term changes in CTC numbers in multiple myeloma and Lewis lung carcinoma xenograft models. We analyzed CTC detections in over 100 h of DiFC data, and considered intervals corresponding to approximately 1%, 5%, 10%, and 20% of the PB volume. In addition, we analyzed changes in CTC numbers over 24 h (diurnal) periods. Results For rare CTCs (fewer than 1 CTC per ml of blood), the use of short DiFC intervals (corresponding to small PB samples) frequently resulted in no detections. For more abundant CTCs, CTC numbers frequently varied by an order of magnitude or more over the time-scales considered. This variance in CTC detections far exceeded that expected by Poisson statistics or by instrument variability. Rather, the data were consistent with significant changes in mean numbers of CTCs on the timescales of minutes and hours. Conclusions The observed temporal changes can be explained by known properties of CTCs, namely, the continuous shedding of CTCs from tumors and the short half-life of CTCs in blood. It follows that the number of cells in a blood sample are strongly impacted by the timing of the draw. The issue is likely to be compounded for multicellular CTC clusters or specific CTC subtypes, which are even more rare than single CTCs. However, we show that enumeration can in principle be improved by averaging multiple samples, analysis of larger volumes, or development of methods for enumeration of CTCs directly in vivo.

Recently, selective inhibition of BET BD2 is emerging as a promising strategy for drug discovery. Despite significant progress in this area, systematic studies of selective BET BD2 inhibitors are still few. In this study, we report the discovery of a potent and selective BET BD2 inhibitor BY27 (47). Our high resolution co-crystal structures of 47/BRD2 BD1 and BD2 showed that the triazole group of 47, water molecules, H433 and N429 in BRD2 BD2 established a water-bridged H-bonding network, which is responsible for the observed selectivities. DNA microarray analysis of HepG2 cells treated with 47 or OTX015 demonstrated the transcriptome impact differences between a BET BD2 selective inhibitor and a pan BET inhibitor. In a MV4-11 mouse xenograft model, 47 caused 67% of tumor growth inhibition and was less toxic than a pan BET inhibitor 1 at high doses. We conclude that the improved safety profile of selective BET BD2 inhibitors warrant future studies in BET associated diseases.

Background With the introduction of targeted therapies, there has been a growing need for non-invasive imaging methods which accurately evaluate therapeutic effects and overcome the limitations of tumor size-based therapeutic response assessments. Purpose To assess diagnostic values of intra-voxel incoherent motion (IVIM) imaging in evaluating therapeutic effects of sorafenib on hepatocellular carcinoma (HCC) using mouse xenograft model. Material and Methods Twenty-four mice bearing Huh-7 were divided into a control group and two treatment groups received sorafenib doses of 5 mg/kg (5 mg-Tx) or 30 mg/kg (30 mg-Tx). IVIM imaging was performed using 10 b-values (0–900 s/mm2). The apparent diffusion coefficient (ADC), diffusion coefficient ( D), and perfusion fraction ( f) were measured for whole tumors and tumor periphery. Changes between baseline and post-treatment parameters ( Δ ADC, Δ D, and Δ f) were calculated, and these parameters were compared with microvessel density (MVD) and area of tumor cell death. Results The post-treatment f and Δ f for tumor periphery were significantly higher in control group, followed by 5 mg-Tx and 30 mg-Tx ( P Conclusion The f is significantly correlated with MVD of HCC, and could potentially be used to evaluate the anti-angiogenic effects of sorafenib.

The orally bioavailable 1-deoxy-sphingosine analog, Enigmol, has demonstrated anticancer activity in numerous in vivo settings. However, as no Enigmol analog with enhanced potency in vitro has been identified, a new strategy to improve efficacy in vivo by increasing tumor uptake was adopted. Herein, synthesis and biological evaluation of two novel fluorinated Enigmol analogs, CF3-Enigmol and CF2-Enigmol, are reported. Each analog was equipotent to Enigmol in vitro, but achieved higher plasma and tissue levels than Enigmol in vivo. Although plasma and tissue exposures were anticipated to trend with fluorine content, CF2-Enigmol absorbed into tissue at strikingly higher concentrations than CF3-Enigmol. Using mouse xenograft models of prostate cancer, we also show that CF3-Enigmol underperformed Enigmol-mediated inhibition of tumor growth and elicited systemic toxicity. By contrast, CF2-Enigmol was not systemically toxic and demonstrated significantly enhanced antitumor activity as compared to Enigmol.

This abstract was withdrawn by the authors.

INTRODUCTION A primary limitation in anti-cancer therapy is the resistance of cancer cells to chemotherapeutic drugs. However, combination therapy may be an effective approach for reducing drug derived toxicity and evading drug resistance, resulting in improved clinical treatment of cancer. Our prior work demonstrated effective treatment of glioblastoma (GBM) with cold atmospheric plasma (CAP) technology with minimal effect to normal cells. Consequently, CAP may serve as a strong candidate for combination therapy with the classical antineoplastic alkylating agent Temozolomide (TMZ) to treat GBM. OBJECTIVES To determine the in vivo co-efficacy of CAP and TMZ to “sensitize” GBM. METHODS An in vivo study was performed using the CAP jet device (He-gas) to determine the effect of combined CAP–TMZ treatment. U87MG-luc glioblastoma cells were implanted intracranially in athymic nude NU(NCr)-Foxn1nu/immunodeficient mice. He-CAP (or control He alone) was non-invasively applied over the skin for 60sec to developed tumors on the first day of the treatment followed with 6.5 mg/kg TMZ or vehicle control treatment for 5 days for two weeks (n=5/group). In vivo bioluminescence imaging was used to monitor tumor volume on the 6th, 9th and 13th treatment day. RESULTS In vivo bioluminescence imaging revealed a marked 8.0±3.2 fold increase in tumor volume in control animals (He-vehicle). Treatment with He-TMZ (6.7±2.5 fold) or CAP-vehicle (4.8±1.7 fold) in isolation had minimal effect in preventing tumor growth. However, combined CAP-TMZ co-treatment virtually prevented increases in tumor volume over 2 weeks (1.8±0.2 fold). CONCLUSIONS Collectively, these findings indicate an effective synergistic treatment method for GBM combining CAP with TMZ. Future investigations look to incorporate radiation into the treatment regimen as well as primary GBM cell models.

Non-small cell lung cancer (NSCLC) accounting for 85 % of all lung cancer was one of the main causes of death worldwide. In this study, we investigated the role of circRNA_102179 in NSCLC development. The levels of circRNA_102179 in NSCLC tissues and cell lines were determined by quantitative real-time PCR assay (qRT-PCR). CCK8 and colony formation assays were applied to explore the effect of circRNA_102179 on the growth of NSCLC cells in vitro. Transwell assay was utilized to analyze the impact of circRNA_102179 on the migration and invasion of NSCLC cells. Target prediction and luciferase reporter assay were used to identify the interacting miRNA of circRNA_102179. The interaction among circRNA_102179/ miR-330-5p/HMGB3 was further validated by colony formation and Transwell invasion assays. Finally, the mouse xenograft NSCLC model was used to explore the role of circRNA_102179 in the tumor growth of NSCLC cells in vivo. CircRNA_102179 was overexpressed in NSCLC tissues and cells compared with normal lung tissues and human bronchial epithelial cells (HBEs). The down-regulation of circRNA_102179 markedly reduced the proliferation, migration, and invasion of NSCLC cells. Moreover, down-expression of circRNA_102179 significantly increased the level of miR-330-5p/HMGB3 in NSCLC cells. Further functional experiments indicated that over-expression of miR-330-5p reversed the inhibitory effect of circRNA_102179 on NSCLC cells growth, migration, and invasion. Our results reveal that circRNA_102179 facilitates the proliferation, migration, and invasion of NSCLC cell via modulating miR-330-5p/ HMGB3 axis in NSCLC cells.

e16750 Background: GP-2250, a novel oxathiazine derivative, displayed apoptotic cytotoxicity against various tumor cell lines but not normal cells. It was therefore tested whether its antineoplastic potential - alone or in combination – could be leveraged specifically against pancreatic cancer. Methods: GP-2250 is a cancer metabolism-based therapeutic. It depleted metabolic energy through inhibition of the enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase) which is rate limiting for aerobic glycolysis. ATP was decreased in a time- and dose-dependent manner in pancreatic tumor cell lines and ROS was increased. Mitochondrial dysfunction was triggered by an increased expression of Bax and decreased expression of Bcl2, leading to apoptosis. Cytotoxicity of GP-2250 was ROS-dependent. It was blocked by N-acetylcysteine. Results: GP-2250 substantially increased the sensitivity of pancreatic tumor cells to various chemotherapeutics in particular to gemcitabine (Gem). At doses which were inactive or barely active per se, the combination of GP-2250 and Gem caused striking cytotoxicity in patient-derived primary tumor cells in vitro, pointing to a strong synergy between the two agents. This finding was substantiated in vivo by patient-derived xenograft (PDX) studies in nude mice. While GP-2250 and Gem, given as monotherapy (500 mg/kg and 50 mg/kg respectively, 2x/week), showed only a limited antineoplastic response, the combination treatment resulted in a significantly higher anti-tumor activity as shown in further PDX. Tumor regression was found in 5 out of 9 PDX based on RECIST criteria. Stable disease was reached in 3 of the remaining grafts. In 1 xenograft, which was unresponsive to Gem, the combination treatment nevertheless achieved a reduction in tumor growth which significantly exceeded that of GP-2250 monotherapy. Conclusions: GP-2250 is a novel cancer metabolism-based therapeutic. GP-2250, in combination with Gem, strongly reduces tumor growth in patient-derived xenografts exceeding by far the response to monotherapy. GP-2250 is being evaluated in a Phase I clinical trial in patients diagnosed with advanced pancreatic cancer (Clinical Trial NCT03854110).

Expression of the receptor tyrosine kinase HER3 is negatively correlated with survival in ovarian cancer, and HER3 overexpression is associated with cancer progression and therapeutic resistance. Thus, improvements in HER3-targeted therapy could lead to significant clinical impact for ovarian cancer patients. Previous work from our group established multivalency as a potential strategy to improve the therapeutic efficacy of HER3-targeted ligands, including affibodies. Others have established HER3 affibodies as viable and potentially superior alternatives to monoclonal antibodies for cancer therapy. Here, bivalent HER3 affibodies were engineered for optimized production, specificity, and function as evaluated in an ovarian cancer xenograft model. Enhanced inhibition of HER3-mediated signaling and increased HER3 downregulation associated with multivalency could be achieved with a simplified construct, potentially increasing translational potential. Additionally, functional effects of affibodies due to multivalency were found to be specific to HER3 targeting, suggesting a unique molecular mechanism. Further, HER3 affibodies demonstrated efficacy in ovarian cancer xenograft mouse models, both as single agents and in combination with carboplatin. Overall, these results reinforce the potential of HER3-targeted affibodies for cancer therapy and establish treatment of ovarian cancer as an application where multivalent HER3 ligands may be useful. Further, this work introduces the potential of HER3 affibodies to be utilized as part of clinically relevant combination therapies (e.g., with carboplatin).

Adult rats were trained to use their forepaw for retrieving a piece of noodle through a slit in the front of the cage. The dorsal corticospinal tract was lesioned by a focal stereotactic radio-frequency lesion at the level of the first/second cervical segment. Complete destruction of one side of the corticospinal tract completely prevented the use of the ipsilateral forepaw reaching for at least 6 months after operation. Rats which have shown no forepaw retrieval by 8 weeks were xenotransplanted with a suspension of cultured olfactory ensheathing cells derived from the mouse olfactory bulb, into the lesion site. Starting between 1 and 3 weeks, 10 rats with transplants bridging the lesion site resumed ipsilateral forepaw reaching. The histology of the lesioned rats with misplaced olfactory ensheathing cell showed no functional recovery during the 8 weeks of training.

The availability of precisely modulated chemical modifications dramatically affects the physicochemical properties of pristine drugs and should facilitate the amphiphilic self-assembly of prodrugs into supramolecular nanoprodrugs (SNPs). However, rationally designing such prodrugs to achieve favorable clinical outcomes still remains a challenge. Here, a library of prodrugs through site-specific attachment of a variety of lipophilic moieties to the antitumor agent SN-38 (7-ethyl-10-hydroxycamptothecin) is constructed. Taking advantage of the role of hydroxyl groups as solvophilic moieties, these prodrugs exhibit self-assembly in aqueous environments, allowing for the identification of five prodrugs capable of self-assembling into SNPs at high drug concentrations. Importantly, in vivo studies demonstrate that the antitumor activity of the SNPs correlates well with their stability and long-term circulation. In addition, the modular feature of this SNP design strategy offers the opportunity to readily incorporate additional valuable functionalities (e.g., tumor-specific targeting ligands) to the particle surface, which is further exploited to improve antitumor efficacy in mouse xenograft models. Thus, this structure-based reconstruction of SN-38 molecules significantly improves the potency of SNPs for clinical use. These results also provide novel mechanistic insights into the rational design of prodrugs.

Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.