The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells.

Osteosarcoma is an aggressive bone cancer affecting both humans and dogs, often leading to pulmonary metastasis. Despite surgery and chemotherapy being the primary treatment modalities, survival rates remain low in both species, underscoring the urgent need for more efficacious therapeutic options. Accumulating evidence indicates numerous biological and clinical similarities between human and canine osteosarcoma, making it an ideal choice for comparative oncological research that should benefit both species. The EphA2 receptor has been implicated in controlling invasive responses across different human malignancies, and its expression is associated with poor prognosis. In this study, we utilized a comparative approach to match EphA2 functions in human and canine osteosarcoma models. Our objectives were to assess EphA2 levels and its pro-malignant action in osteosarcoma cells of both species. We found that EphA2 is overexpressed in most of both canine and human osteosarcoma cell lines, while its silencing significantly reduced cell viability, migration, and invasion. Moreover, EphA2 silencing enhanced the sensitivity of osteosarcoma cells to cisplatin, a drug commonly used for treating this cancer. Furthermore, inhibition of EphA2 expression led to a significant reduction in tumor development capability of canine osteosarcoma cells. Our data suggest that these EphA2 effects are likely mediated through various signaling mechanisms, including the SRC, AKT, and ERK-MAPK pathways. Collectively, our findings indicate that EphA2 promotes malignant behaviors in both human and canine osteosarcoma and that targeting EphA2, either alone or in combination with chemotherapy, could offer potential benefits to osteosarcoma patients.

A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2-Bispecific Antibody for Combination Cancer Therapy.

PURPOSE: Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics. EXPERIMENTAL DESIGN: Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models. RESULTS: Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab. CONCLUSIONS: Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment. See related commentary by Kumar, p. 2570.

A Systematic Investigation into the Influence of Net Charge on the Biological Distribution of Radiometalated Peptides Using [68Ga]Ga-DOTA-TATE Derivatives.

Recently, several radiometalated peptides have been approved for clinical imaging and/or therapy (theranostics) of several types of cancer; nonetheless, the primary challenge that most of these peptides confront is significant renal uptake and retention, which is often dose limiting and can cause nephrotoxicity. In response to this, numerous methods have been employed to reduce the uptake of radiometalated peptides in the kidneys, and among these is adding a linker to modulate polarity and/or charge. To better understand the influence of net charge on the biodistribution of radiometalated peptides, we selected the clinically popular construct DOTA-TATE (NETSPOT/LUTATHERA) as a model system. We synthesized derivatives using manual solid-phase peptide synthesis methods including mechanical and ultrasonic agitation to effectively yield the gold standard DOTA-TATE and a series of derivatives with different net charges (+2, +1, 0, -1, -2). Dynamic PET imaging from 0 to 90 min in healthy female mice (CD1) revealed high accumulation and retention of activity in the kidneys for the net-neutral (0) charged [68Ga]Ga-DOTA-TATE and even higher for positively charged derivatives, whereas negatively charged derivatives exhibited low accumulation and fast renal excretion. Ex vivo biodistribution at 2 h post injection demonstrated a significant retention of [68Ga]Ga-DOTA-TATE (∼74 %ID/g) in the kidneys, which increased as the net positive charge per molecule increased to +1 and +2 (∼272 %ID/g and ∼333 %ID/g, respectively), but the -1 and -2 net charged molecules exhibited lower renal uptake (∼15 %ID/g and 16 %ID/g, respectively). Interestingly, the net -2 charged [68Ga]Ga-DOTA-(Glu)2-PEG4-TATE was stable in blood serum but had much higher healthy organ uptake (lungs, liver, spleen) than the net -1 compound, suggesting instability in vivo. Although the [68Ga]Ga-DOTA-PEG4-TATE derivative with a net charge of 0 also showed a decrease in kidney uptake, it also showed instability in blood serum and in vivo. Despite the superior pharmacokinetics of the net -1 charged [68Ga]Ga-DOTA-Glu-PEG4-TATE in healthy mice with respect to kidney uptake and overall profile, dynamic PET images and ex vivo biodistribution in male mice (NSG) bearing AR42J (SSTR2 overexpressing) subcutaneous tumor xenografts showed significantly diminished tumor uptake when compared to the gold standard [68Ga]Ga-DOTA-TATE. Taken together, these findings indicate unambiguously that kidney uptake and retention are significantly influenced by the net charge of peptide-based radiotracers. In addition, it was illustrated that the negatively charged peptides had substantially decreased kidney uptake, but in this instantiation the tumor uptake was also impaired.