Ying Zhang

The manuscript entitled “TARGETING ALZHEIMER'S DISEASE HALLMARKS WITH THE NRF2 ACTIVATOR ISOEUGENOL” related to Alzheimer's disease, including potential therapeutic interventions, regulatory aspects et.al. It discusses the potential therapeutic effec...

Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein–engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.

Surface-exposed calreticulin (ecto-CRT) is a well-known “eat-me” signal exhibited by dying cells that contributes to their recognition and destruction by the immune system. We assessed the use of a CRT-specific binding peptide for imaging ecto-CRT during immunogenic cell death and its utility for early prediction of treatment response. Methods: A synthetic CRT-specific peptide, KLGFFKR (CRTpep), was labeled with fluorescein isothiocyanate or ¹⁸F, and the characteristics of ecto-CRT were evaluated in a colon cancer cell line in vitro and in vivo. Results: In vitro flow cytometry, immunofluorescence staining, and in vivo small-animal PET imaging results showed that CRTpep detected preapoptotic cells treated with immunogenic drugs or radiation but not those treated with the nonimmunogenic drug or a nontherapeutic dose of immunogenic drug. Conclusion: The present results indicate that the CRT-specific peptide would enable the prediction of therapeutic response, thereby facilitating early decisions on continuation or discontinuation of immunogenic treatment. Visual Abstract

Tumor-targeting bacteria have been actively investigated as a new therapeutic tool for solid tumors. However, in vivo imaging of tumor-targeting bacteria has not been fully established. ¹⁸F-fluorodeoxysorbitol (FDS) positron emission tomography (PET) is known to be capable of imaging Gram-negative Enterobacteriaceae infection. In the present study, we aimed to validate the use of ¹⁸F-FDS PET for visualization of the colonization and proliferation of tumor-targeting Escherichia coli (E. coli) MG1655 in mouse tumor models.

Photoacoustic imaging (PAI) is a new and attractive imaging modality, and it has strong potential for application in the early detection of tumors through the use of optically absorbing targeted contrast agents. Ag₂S quantum dots (QD) are a promising bionanomaterial and have attracted significant attention in the field of bioimaging. In this study, water-soluble and carboxylic acid group-coated Ag₂S QDs with an ultrasmall size (∼8 nm) were synthesized via a one-step method. Their surface plasmon resonance wavelength was determined to be ∼800 nm, which is ideal for PAI. Ag₂S QDs were then modified with the epidermal growth factor receptor 1 (EGFR) targeted small protein affibody Z_EGFR:1907. The resulted nanoprobe, Z_EGFR:1907-Ag₂S QDs, was then used for targeted PAI of EGFR-overexpressed tumors. The biodistribution of the nanoprobe was further measured by ex vivo near infrared fluorescence (NIRF) imaging of the dissected tissues. The PAI results showed that Z_EGFR:1907-Ag₂S QDs specifically image EGFR positive tumors. The biodistribution study revealed that the nanoprobe mainly accumulated in the liver, spleen and tumors; tissue H&E staining studies indicated that the probe has good biocompatibility. Overall, the affibody-functionalized Ag₂S QDs are a novel targeted nanoprobe that can be used for specific PAI of tumors.