Marine natural product lepadin A as a novel inducer of immunogenic cell death via CD91-dependent pathway

Immunogenic Cell Death(ICD)represents a mechanism of enhancing T cell-driven response against tumor cells.The process is enabled by release of damage-associated molecular patterns(DAMPs)and cytokines by dying cells.Based on molecular studies and clinical marker assessment,ICD can be a new target for cancer chemotherapy hitherto restricted to a few conventional anticancer drugs.In view of the development of small molecules in targeted cancer therapy,we reported the preliminary evidence on the role of the natural product lepadin A(1)as a novel ICD inducer.Here we describe the ICD mechanism of lepadin A(1)by proving the translocation of the protein calreticulin(CRT)to the plasma membrane of human A2058 melanoma cells.CRT exposure is an ICD marker in clinical studies and was associated with the activation of the intrinsic apoptotic pathway in A2058 cells with lepadin A(1).After the treatment,the tumour cells acquired the ability to activate dendritic cells(DCs)with cytokine release and costimulatory molecule expression that is consistent with a phenotypic profile committed to priming T lymphocytes via a CD91-dependent mechanism.The effect of lepadin A(1)was dose-dependent and comparable to the response of the chemotherapy drug doxorubicin(2),a well-established ICD inducer.

Tomographic flow cytometry by digital holography

High-throughput single-cell analysis is a challenging task.Label-free tomographic phase microscopy is an excellent candidate to perform this task.However,in-line tomography is very difficult to implement in practice because it requires a complex set-up for rotating the sample and examining the cell along several directions.We demonstrate that by exploiting the random rolling of cells while they are flowing along a microfluidic channel,it is possible to obtain in-line phase-contrast tomography,if smart strategies for wavefront analysis are adopted.In fact,surprisingly,a priori knowledge of the three-dimensional position and orientation of rotating cells is no longer needed because this information can be completely retrieved through digital holography wavefront numerical analysis.This approach makes continuous-flow cytotomography suitable for practical operation in real-world,single-cell analysis and with a substantial simplification of the optical system;that is,no mechanical scanning or multi-direction probing is required.A demonstration is given for two completely different classes of biosamples:red blood cells and diatom algae.An accurate characterization of both types of cells is reported,despite their very different nature and material content,thus showing that the proposed method can be extended by adopting two alternate strategies of wavefront analysis to many classes of cells.