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Simple Summary Lung cancer is often detected at late stages when metastases are present and the genomic make-ups of the tumors are heterogeneous. Analyses of genomic alterations in non-small-cell lung cancer (NSCLC) have revealed mutated tumor-associated membrane receptors and fusion proteins, which can be targeted via tyrosine kinase inhibitors (TKIs). TKIs initially often have a good effect, but a fraction of the tumor lesions may develop resistance through additional mutations in the targeted kinases or by increased expression/function of other membrane receptors. Detection of TKI-bypassing mechanisms is difficult in tissue biopsies as these analyze only a subpart of tumors or lesions. Liquid biopsies based on tumor-secreted small extracellular vesicles (sEVs) into body fluids can assess tumor heterogeneity. We present an immuno-PCR method for the detection of the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2), and the insulin-like growth factor 1 receptor (IGF-1R) on sEVs. Initial investigations of sEVs from EGFR-mutant NSCLC tumor cells or pleural effusion (PE) fluid from patients with NSCLC or benign diseases showed different protein profiles for individual sEV samples. Further development of the immuno-PCR could complement DNA/mRNA-based assays detecting kinase mutations to allow longitudinal treatment monitoring of diverse TKI-bypassing mechanisms. Abstract Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an EGFR-driven NSCLC adenocarcinoma than in sEVs from PE samples of non-EGFR driven adenocarcinoma patients or in samples from patients with benign lung disease. In summary, we have developed a diagnostic method for sEVs in liquid biopsies of cancer patients which may be used for longitudinal treatment monitoring to detect emerging bypassing resistance mechanisms in a noninvasive way.

Ephrin (EFN)/ Erythropoietin-producing human hepatocellular receptors (Eph) signaling has earlier been reported to regulate non-small cell lung cancer (NSCLC) cell survival and cell death as well as invasion and migration. Here, the role of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) signaling and ionizing radiation (IR) cellular effect was studied in NSCLC cells. Silencing of EphA2 resulted in IR sensitization, with increased activation of caspase-3, PARP-1 cleavage and reduced clonogenic survival. Profiling of EphA2 expression in a NSCLC cell line panel showed a correlation to an IR refractory phenotype. EphA2 was found to be transiently and rapidly phosphorylated at Ser897 in response to IR, which was paralleled with the activation of ribosomal protein S6 kinase (RSK). Using cell fractionation, a transient increase in both total and pSer897 EphA2 in the nuclear fraction in response to IR was revealed. By immunoprecipitation and LC-MS/MS analysis of EphA2 complexes, nuclear localized EphA2 was found in a complex with DNA-PKcs. Such complex formation rapidly increased after IR but returned back to basal level within an hour. Targeting EphA2 with siRNA or by treatment with EFNA1 ligand partly reduced phosphorylation of DNA-PKcs at S2056 at early time points after IR. Thus, we report that EphA2 interacts with DNA-PKcs in the cell nucleus suggesting a novel mechanism involving the EphA2 receptor in DDR signaling and IR responsiveness.

RT treatment schedule and transducer replacement schedule. Rats received RT on days 0-4 and 7-11 (indicated by V). Groups 4 and 5 had arrays placed on their dorsal surface (arrays where replaced days indicated by V). Rats where euthanized on day 12. (DOCX 96 kb)

TTFields Delay Irradiation-Induced DNA Damage Repair in glioma cells. LN-18 cells were irradiated with 4 Gy RT and immediately treated with TTFields applied for 1 h, 2 h or 24 h (A-B). Effect on DNA repair was measured as tail moment in the comet assay. (PPTX 1940 kb)

TTFields effect on LN-18 glioma cells. Surviving fractions of LN-18 cells treated with TTFields (200 kHz, 1.0 V/cm) for 72 h either alone or immediately after irradiation with 4 Gy (A). Surviving fraction of LN-18 cells treated with RT alone or with RT at various doses followed by 200 kHz TTFields (1.0 V/cm RMS) for 72 h. Results of the combined treatments were normalized to the effect of TTFields alone (B). (PPTX 2783 kb)