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A major barrier to the successful application of nanotechnology for cancer treatment is the suboptimal delivery of therapeutic payloads to metastatic tumor deposits. We previously discovered that cabozantinib, a tyrosine kinase inhibitor, triggers neutrophil-mediated anticancer innate immunity, resulting in tumor regression in an aggressive PTEN/p53-deficient genetically engineered murine model of advanced prostate cancer. Here, we specifically investigated the potential of cabozantinib-induced neutrophil activation and recruitment to enhance delivery of BSA-coated polymeric nanoparticles (BSA-NPs) into murine PTEN/p53-deficient prostate tumors. On the basis of the observation that BSA coating of NPs enhanced association and internalization by activated neutrophils by approximately 6-fold in vitro, relative to uncoated NPs, we systemically injected BSA-coated, dye-loaded NPs into prostate-specific PTEN/p53-deficient mice that were pretreated with cabozantinib. Flow cytometric analysis revealed an approximately 4-fold increase of neutrophil-associated BSA-NPs and an approximately 32-fold increase in mean fluorescent dye uptake following 3 days of cabozantinib/BSA-NP administration, relative to BSA-NP alone. Strikingly, neutrophil depletion with Ly6G antibody abolished dye-loaded BSA-NP accumulation within tumors to baseline levels, demonstrating targeted neutrophil-mediated intratumoral NP delivery. Furthermore, we observed an approximately 13-fold decrease in accumulation of BSA-NPs in the liver, relative to uncoated NPs, post-cabozantinib treatment, suggesting that BSA coating of NPs can significantly enhance cabozantinib-induced, neutrophil-mediated targeted intratumoral drug delivery, while mitigating off-target toxicity. Collectively, we demonstrate a novel targeted nano-immunotherapeutic strategy for enhanced intratumoral delivery of BSA-NPs, with translational potential to significantly augment therapeutic indices of cancer medicines, thereby overcoming current pharmacologic barriers commonly encountered in preclinical/early-phase drug development.

Cancer metastasis is a critical culprit frequently blamed for treatment failure, drug resistance, poor prognosis, and high mortality rate among all human cancers. Laboratory efforts to isolate metastatic cell populations have typically been confined to mouse models, which are time-consuming and expensive. Here, we present a model system based on xenografting zebrafish embryos to select for cells that are predisposed to progress through the early stages of metastasis. This model requires only 3-5 days to achieve distinct intravasation to the zebrafish circulatory system. The metastatic cells are easily tracked in real-time as they migrate, as well as isolated and propagated in vitro. Once expanded, molecular characterization of the serially derived invasive cell populations from the tails of the zebrafish accurately predicts genes, signaling pathways, protein-protein interactions, and differential splicing products that are important for an invasive phenotype. This zebrafish model therefore offers a high-throughput and robust method for identifying gene targets critical for cancer metastasis.

A major barrier to the successful application of nanotechnology for cancer treatment is the efficient delivery of therapeutic payloads to metastatic tumor deposits. We have previously discovered that cabozantinib, a tyrosine kinase inhibitor, triggers neutrophil-mediated anti-cancer innate immunity, resulting in tumor regression in an aggressive PTEN/p53-deficient genetically engineered murine model of advanced prostate cancer. Here, we specifically investigated the potential of cabozantinib-induced neutrophil activation and recruitment to enhance delivery of bovine serum albumin (BSA)-coated polymeric nanoparticles (NPs) into murine PTEN/p53-deficient prostate tumors. Based on the observation that BSA-coating of NPs enhanced association and internalization by activated neutrophils in vitro, relative to uncoated NPs, we systemically injected BSA-coated, dye-loaded NPs into prostate-specific PTEN/p53-deficient mice that were pre-treated with cabozantinib. Flow cytometric analysis revealed a 4-fold increase of neutrophil-associated NPs within the tumor microenvironment (TME) of mice pre-treated with cabozantinib relative to untreated controls. At steady-state, following 3 days of cabozantinib/NP administration, 1% of systemically injected dye-loaded NPs selectively accumulated within the TME of mice that were pre-treated with cabozantinib, compared to 0.11% uptake for mice that did not receive cabozantinib pre-treatment. Strikingly, neutrophil depletion with Ly6G antibody abolished NP accumulation in tumors to baseline levels, demonstrating targeted neutrophil-mediated NP delivery to the prostate TME. In summary, we have discovered a novel nano-immunotherapeutic strategy for enhanced intratumoral delivery of injected NPs, which results in significantly higher NP accumulation than reported strategies in the nanotechnology literature to-date.

Nature Communications 8: Article number: 16081 (2017); Published: 17 July 2017, Updated: 13 July 2018 The original version of this Article omitted the following from the Acknowledgements: ‘We thank Robert Kirkpatrick for implementing the high throughput protein design strategy that enabled screeningand triage of essential A.

Interest in the field of antibody—drug conjugates (ADCs) has grown exponentially over the past decade. As the product pipeline grows, there is increasing need for robust chemistries which allow selective and efficient functionalization of the antibody cores for introduction of appropriate linkers and spacer groups. Under conventional bioconjugation conditions, product heterogeneity often results, and the drug-to-antibody ratios (DAR) are inconsistent. Based on our experience in protein derivatization, we have investigated the potential for continuous-flow microreactor technology to expedite and facilitate such processes. We demonstrate its potential using reagent proteins and the chimeric monoclonal antibody infliximab (Remicade™).

Adenoid cystic carcinomas (ACC) are rare salivary gland cancers with a high incidence of metastases. In order to study this tumor type, a reliable model system exhibiting the molecular features of this tumor is critical, but none exists, thereby inhibiting in-vitro studies and the analysis of metastatic behavior. To address this deficiency, we have coupled an efficient method to establish tumor cell cultures, conditional reprogramming (CR), with a rapid, reproducible and robust in-vivo zebrafish model. We have established cell cultures from two individual ACC PDX tumors that maintain the characteristic MYB translocation. Additional mutations found in one ACC culture also seen in the PDX tumor. Finally, the CR/zebrafish model mirrors the PDX mouse model and identifies regorafenib as a potential therapeutic drug to treat this cancer type that mimic the drug sensitivity profile in PDX model, further confirming the unique advantages of multiplex system.

A facile and benign route to N-heterocycles, including triazoles and triazolopyrimidines, has been developed. Using continuous-flow microreactor technology, organic azides are prepared in situ and reacted with cyanoacetamide in a [3+2] cycloaddition to produce a variety of substituted 1,2,3-triazoles, which can be elaborated into useful building blocks. A benzyl-substituted triazole was further functionalized to an analog of the core structure of the antiplatelet agent Brilinta®. The methodology lends itself well to flow chemistry, where reaction volumes are minimized, heating and mixing are consistent, and the need for intermediate azide isolation bypassed. The scope of the process is wide, and the efficiency is high, suggesting this as a practical, green route for the production of triazolo-based heterocycles.

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery., Encoded Library Technology (ELT) has streamlined the identification of chemical ligands for protein targets in drug discovery. Here, the authors optimize the ELT approach to screen multiple proteins in parallel and identify promising targets and antibacterial compounds for S. aureus, A. baumannii and M. tuberculosis.

Purpose/Objectives: The lack of authenticated cell lines from rare tumors, such as adenoid cystic carcinoma (ACC), have compromised studies on basic biology and drug development. ACC of the salivary gland is a rare human cancer and is classified as a rare or “orphan” disease. It has the propensity for invasion of peripheral nerves or blood vessels early in the disease course resulting in a high incidence of both local and distant metastases to lung and bone. The primary course of treatment is surgical removal of tumor, which is usually combined with postoperative radiotherapy. To date no targeted therapy exists for this tumor type. Patient-derived mouse xenografts (PDX) have been shown to maintain the histology and gene expression profile of the primary tumor, making them a valid model system. However, PDX models suffer from high cost, expensive maintenance, low take rate (30-50%), lack of manipulation and high throughput capability. In order to study these tumor types, a reliable model system is critical that can maintain the key genetic features of the primary tumor. Materials/Methods: A new cell culture method, conditional reprogramming (CR), combines the use of irradiated mouse fibroblasts and a ROCK inhibitor to induce the rapid, efficient, and long-term growth of normal and tumor cells without requiring exogenous viral or cellular gene expression. We used this technology to establish cell cultures from rare tumor types such as ACC, neuroendocrine, anal cancer and recurrent respiratory papillomatosis. Herein, we present data collected on recently established stable cultures for ACC using PDX tissue materials. We tested the maintenance of metastasis in the cell lines by establishing a rapid and robust in-vivo zebrafish xenograft model system for ACCs, using ACC11 as a prototype. Results: We generated stable cultures for ACC from eight individual PDX tumors using CRC technology. We demonstrate that these cultures maintain the key genetic features of the primary tumors. One ACC cell line, ACC11, has shown to maintain the MYB-NFIB translocation, oncogenic mutations in FGFR2 and ATM genes and overexpression of Myb-NF1B fusion protein similar to the tumor of origin (PDX tumor). In the zebrafish in-vivo system, these cells are migratory and possess metastatic potential demonstrated by migration of tumor cells from the 2-day post fertilized yolk sac to the tail including vascular invasion within 3 days post injection. Similar results were obtained when PDX tissue material for ACC11 as well as a conventional metastatic breast cancer cell line, MDA-MB231. In contrast, injection of a patient-derived normal breast cell line established using the CR technology and a conventional non-metastatic breast cancer cell line, MCF7, did not show any movement of cells even after 7 days of post injection. Conclusions: We have successfully established a tool-kit consisting of an in-vitro (cell line) and a rapid in-vivo (zebrafish) metastasis model system for biological and translational research of rare tumor types. The use of this novel CR method has great potential in rapidly generating patient-derived normal and cancer primary cells for a wide variety of uses including biobanking, basic tumor biology, drug target identification, and drug discovery, with an ultimate goal of personalized cancer medicine. This abstract is also being presented as Poster B27. Citation Format: Chen Chen, Sujata Choudhury, Devan Wilkins, Eric Glasgow, Richard Schlegel, Seema Agarwal. Conditional reprogramming is successful in generating patient-derived stable cell cultures from rare aggressive tumors. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr PR06.