Chemotherapeutic drugs suffer from non-specific binding, undesired toxicity, and poor blood circulation which contribute to poor therapeutic efficacy. In this study, antibody–drug nanoparticles (ADNs) are engineered by synthesizing pure anti-cancer drug nanorods (NRs) in the core of nanoparticles with a therapeutic monoclonal antibody, Trastuzumab on the surface of NRs for specific targeting and synergistic treatments of human epidermal growth factor receptor 2 (HER2) positive breast cancer cells. ADNs were designed by first synthesizing ~ 95 nm diameter × ~ 500 nm long paclitaxel (PTX) NRs using the nanoprecipitation method. The surface of PTXNRs was functionalized at 2′ OH nucleophilic site using carbonyldiimidazole and conjugated to TTZ through the lysine residue interaction forming PTXNR-TTZ conjugates (ADNs). The size, shape, and surface charge of ADNs were characterized using scanning electron microscopy (SEM), SEM, and zeta potential, respectively. Using fluorophore labeling and response surface analysis, the percentage conjugation efficiency was found > 95% with a PTX to TTZ mass ratio of 4 (molar ratio ≈ 682). In vitro therapeutic efficiency of PTXNR-TTZ was evaluated in two HER2 positive breast cancer cell lines: BT-474 and SK-BR-3, and a HER2 negative MDA-MB-231 breast cancer cell using MTT assay. PTXNR-TTZ inhibited > 80% of BT-474 and SK-BR-3 cells at a higher efficiency than individual PTX and TTZ treatments alone after 72 h. A combination index analysis indicated a synergistic combination of PTXNR-TTZ compared with the doses of single-drug treatment. Relatively lower cytotoxicity was observed in MCF-10A human breast epithelial cell control. The molecular mechanisms of PTXNR-TTZ were investigated using cell cycle and Western blot analyses. The cell cycle analysis showed PTXNR-TTZ arrested > 80% of BT-474 breast cancer cells in the G2/M phase, while > 70% of untreated cells were found in the G0/G1 phase indicating that G2/M arrest induced apoptosis. A similar percentage of G2/M arrested cells was found to induce caspase-dependent apoptosis in PTXNR-TTZ treated BT-474 cells as revealed using Western blot analysis. PTXNR-TTZ treated BT-474 cells showed ~ 1.3, 1.4, and 1.6-fold higher expressions of cleaved caspase-9, cytochrome C, and cleaved caspase-3, respectively than untreated cells, indicating up-regulation of caspase-dependent activation of apoptotic pathways. The PTXNR-TTZ ADN represents a novel nanoparticle design that holds promise for targeted and efficient anti-cancer therapy by selective targeting and cancer cell death via apoptosis and mitotic cell cycle arrest.

Programmed cell death, or apoptosis, and controlled cell division, or mitosis, are two highly regulated processes in the cell cycle. A balance between apoptosis and mitosis is critical for multiple distinct states including embryonic development, immune cell activation, stem cell differentiation, tissue formation (wound healing), and tumor prevention, among others. A cell undergoing apoptosis shows a series of characteristic morphological changes similar to normal mitosis and an aberrant form of mitosis. During each of these processes, nuclear chromatin condenses, the nuclear lamina and cytoplasmic membranes disintegrate, and cells decrease in volume. The morphological resemblance among cells undergoing these processes suggests that the underlying intracellular signaling pathways influence the mitotic cell fate. In this paper, the relationship of intracellular signaling pathways, cell cycle dynamics, and apoptotic cell signaling pathways is discussed. The mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/Ras/Raf/ERK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), Janus kinase/signal transducer and activator of transcription (JAK/STAT), wingless-related integration site (Wnt), and transforming growth factor beta (TGF-[Formula: see text] are major cell signaling pathways that transmit signals from multiple cell surface receptors to transcription factors in the nucleus. The pathways are stimulated by cytokines, growth factors, and external stimuli, i.e., reactive oxygen species which induce signal transduction pathways and regulate complex processes such as cell cycle progression, cell proliferation, cellular growth, differentiation, and apoptosis. Aberrant mutations in particular genes and proteins of these pathways contribute to cancers usually by inhibiting pro-apoptotic proteins (e.g., Bak, Bax, Noxa, Puma, etc.) and stimulating antiapoptotic proteins (e.g., Bcl-2, Bcl-XL, Mcl-1, etc.). The cell cycle is regulated by intracellular signaling pathways such as the MAPK/Ras/Raf/ERK and PI3K pathways to produce the synthesis of cyclin D and other mitosis regulating proteins (Myc and Jun). Cyclin D1 binds to cyclin-dependent kinase (CDK) 4 and CDK 6 (CDK4/6) to form an effective complex, activate several substrates, and initiate the cell cycle. The prominent molecules that regulate signaling pathways in normal and cancer cells are described.

Aurora kinases (Aur) are a family of serine/threonine kinases essential for genetic stability and cell division during mitosis. In humans, activity of the three paralogs (AurA, AurB, and AurC) is regulated throughout the cell cycle, with peak activity during mitosis. Each paralog shares a conserved C-terminal catalytic domain, but differs in substrate specificity, sub-cellular localization, and function. AurA mainly controls centrosome maturation and bipolar spindle assembly; AurB phosphorylates histone H3 (Ser10), which aids in chromatin condensation and separation; AurC, whose expression is restricted to germ cells of both genders, shares similar characteristics to AurB including substrate localization, specificity, and function during mitosis. Aberrant Aur expression leads to genomic instability or aneuploidy, and overexpression of the three paralogs is found in several cancer types, including lung, colon, and gliomas. Aur inhibition induces growth arrest and apoptosis in a variety of cancer cell types. Thus, design and development of Aur inhibitors as anti-tumor agents has been widely explored in recent years, and many are under preclinical and clinical investigation. We used the Eurofins Discovery OncoPanel™ cell-based profiling platform to investigate the specific effects of AurA, AurB, and AurC inhibitors against a select panel of human cancer cell lines. To determine the mechanistic effect of Aur inhibition in cells, we measured phospho-histone H3 (Ser10) by high-content analysis. OncoPanel human tumor cell lines arrested with nocodazole were treated with Aur inhibitors for 2 hours. Phospho-histone H3 (Ser10) levels were measured and the percentage of the DMSO vehicle-treated control cell signal was calculated for each test point. Ten-point dose-response curves were fitted using a 4-parameter log-logistic model with custom curve-fitting software to determine IC50 values. The inhibitors were also evaluated for their effect on tumor cell proliferation, apoptosis, and cell cycle arrest over the same dose-response range, after a 10-day incubation. Following exposure, the cells were fixed, stained with DAPI, anti-cleaved caspase-3, and anti-phospho-histone H3 (Ser28), and imaged. Cell proliferation dose-response curves were also fitted using the same model as above. Correlations were then made between observed mechanistic and functional activity. Activity of the AurA-selective inhibitor, MLN8054, was also evaluated in the Eurofins Discovery BioMAP® Diversity PLUS human primary cell phenotypic profiling service to give a broader view of its biological activity. These studies demonstrate the combined value of using the OncoPanel and BioMAP platforms to determine mechanistic, functional, and broader biological activity of therapeutic candidates in relevant cellular models for a better translational understanding of potential therapeutic capabilities and accelerate decision-making. Citation Format: Luciano Galdieri, Justin Lipner, Steven M. Garner, Jillian Krings, Emily Hoehn, Brendan Lahm, Kaleb Collver, Kaitlyne Powers, Alastair J. King. Phenotypic profiling of Aurora inhibitors using the OncoPanelTMplatform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2770.

It is estimated that in the U.S. alone, roughly $200 billion are spent each year on cancer drug discovery. Although this spending is significant, we have still to find truly resilient and enduring treatment plans for a significant percentage of human cancers. Approaches to better understand the broader efficacy of cancer therapeutics and the underlying genomics that govern sensitivity have dramatically changed over the years, as has our ability to screen and characterize the drug response of larger numbers of cancer types. There is thus an already significant need for more robust and effective therapeutic agent screening and profiling in drug discovery. Each cancer patient effectively presents a unique disease, which showcases the challenge in finding the most effective cancer treatments for as many patients as possible.The OncoPanel™ cell-based profiling and screening service from Eurofins Discovery generates drug response data across a panel of over 300 genomically diverse human tumor cell lines that span 19 different tissue types. The data generated from such profiling can also enable the identification of potentially predictive genomic biomarkers of drug response. This can in turn help to determine which genomic features may predispose patients to a more beneficial therapeutic response, or identify potential intrinsic resistance mechanisms to new therapeutic agents. The OncoPanel platform provides an opportunity to evaluate cancer therapeutics using a variety of assay formats and exposure times. Our singleplex assay demonstrates the effect of test agents on tumor cell growth, while our mulitplexed assay uses high content analysis to demonstrate in vitro efficacy in terms of cell proliferation, induction of apoptosis, and effects on the cell cycle. This yields valuable information on how therapeutics inhibit human tumor cell growth. However, the effect of therapeutics on cell viability can also be evaluated using standard CellTiter-Glo® format assays. All of these assays can be used with standard exposure times ranging from three to ten days, to accommodate testing of a wide range of therapeutics and target-based modalities. Here we show how we have qualified more human tumor cell lines from a range of different tissue types for addition to the OncoPanel service in the above-mentioned assay formats. This further expands and diversifies the tumor types represented by this platform, which in turn increases our ability to profile therapeutic agents in a wider panel of individual tumor types. Using this expanded cell line panel, we are now able to demonstrate greater breadth of activity of potential anti-cancer drugs in discovery and development for the treatment of a larger range of patients. Citation Format: Steven M. Garner, Jillian V. Krings, Emily C. Hoehn, Erin N. Lofton, Lyndsey M. Rose, Lee R. Cavedine, Brendan M. Lahm, Kaleb Collver, Kaitlyne Powers, Lindsey Herishen, Alastair J. King. Qualification of human tumor cell lines for inclusion in the OncoPanel™ cell-based profiling and screening service [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2769.

Background: Alterations in tumor cell metabolism are one of the central processes guiding cancer progression. The increased glucose dependence of cancer cells, also known as the Warburg effect, opens the possibility to therapeutically target the glycolytic pathway and challenge the metabolic needs of tumor cells. We generated 24 monoclonal antibodies targeting the main glucose transporter on cancer cells - SLC2A1 (GLUT1). We evaluated the ability of the antibodies to affect cancer cell fitness alone and in combination with OXPHOS inhibitors to find clinically applicable therapeutics targeting tumor metabolism. virus like particle (VLP)-based immunization strategy (Kivi, et al., BMC Biotechnology, 2016). Antibodies were functionally characterized by 2-deoxyglucose uptake interference assay and sensitivity to antibody single and combination treatments were evaluated in various tumor cell lines. Metabolomics and oxygen consumption rates were examined as mechanistic endpoints for tumor cell lines and primary tumor samples. Results: The anti-SLC2A1 antibodies specifically bind to SLC2A1 with low nanomolar EC50 values and not to other glucose transporters. Antibody clones with functional properties inhibit glucose uptake leading to reduced metabolic activity and growth inhibition in a subset of cancer cell lines. A drastic cell proliferation inhibition is moreover observed in combination of anti-SLC2A1 antibodies and OXPHOS inhibitors metformin, phenformin or IACS-010759 in 2D cultures of colon, breast and pancreatic cancer cell lines. The inhibition of metabolic activity was further confirmed in an ex vivo primary patient tissue samples highlighting the putative translational applicability of our antibodies. Conclusions: This is the first study to provide highly specific antibodies bocking the function of SLC2A1 transporter and demonstrate the proof of principle for inhibiting complex muti-pass membrane transporters with antibody therapeutics. While the developed anti-SLC2A1 antibodies could be efficacious in some indications as single agents, appreciable clinical activity could be obtained by the combined use with OXPHOS inhibitors. Citation Format: Siret Tahk, Kai Virumäe, Paule Hermet, Korneelia Anton, Maiken Abel, Denis Belitškin, Luciano Galdieri, Steven Garner, Jillian Krings, Kaleb Collver, Emily Hoehn, Brendan Lahm, Kaitlyne Powers, Tuuli Käämbre, Alastair J. King, Francisca Neethling, Anu Planken, Mart Ustav, Andres Männik, Mart Ustav. Functional antibodies against multi span transmembrane proteins - revisiting the Warburg effect in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6036.