A fluorescent biosensor-based platform for the discovery of immunogenic cancer cell death inducers

Abstract

Systemic anticancer immunity can be reinstated via the induction of immunogenic cell death (ICD) in malignant cells. Thus, certain classes of cytotoxic compounds, for example, anthracyclines, oxaliplatin and taxanes are endowed with the capacity to act on cancer cells to ignite premortem stress pathways that lead to the surface exposure of calreticulin (CALR) and the cellular release of adenosine triphosphate, annexin A1, high mobility group B1 and type-1 interferons. Altogether, these alterations constitute the hallmarks of ICD. Here we report the design of a discovery pipeline for the identification of novel ICD inducers by means of a phenotypic screening platform. The use of fluorescent biosensors as proxies for the manifestation of ICD hallmarks has enabled the exploration of large collections of chemical compounds by automatized screening routines. Imaging-based assessment and phenotypic selection led to the identification of potential ICD inducers that could be validated further in vitro and in vivo, confirming that bona fide ICD inducers possess the capacity to induce immunological long-term memory and to confer resistance against rechallenge with syngeneic tumors. Machine learning algorithms analyzing the physicochemical properties of ICD inducers can assist in the preselection of compounds with potential ICD-stimulatory properties, further accelerating the screening efforts designed to develop new immunotherapeutic agents. ARTICLE HISTORY

Detection of immunogenic cell death and its relevance for cancer therapy

“…In summary, we and others have developed assays to assess ICD parameters in vitro (in cell cultures) and ex vivo (on tumor biopsies), which can be harnessed for the discovery of next-generation anticancer agents and the identification of optimal therapeutic regimens for clinical application, respectively. The construction of a multistep discovery pipeline involving artificial intelligence-driven pre-selection and a robotized workflow for the detection of surrogate ICD biomarkers enables us to implement various HCS campaigns that ultimately identified novel ICD inducers 206 . Some of the compounds identified with this platform have entered clinical trials, either as single agents or in combination with ICIs 61 , 63 .…”

“…2 ). Specifically, we employ biosensor cell lines to measure fluorescent surrogate markers for ATP release (with quinacrine), CALR exposure (using cells stably expressing CALR-GFP), type I IFN signaling (with cells expressing GFP under the control of the MX1 promoter), and HMGB1 release (in cells stably transduced with an HMGB1-GFP fusion) along with morphological traits of cell death such as the rarefaction of cells or the appearance of pyknotic nuclei 206 . These biosensors can be cultured in the presence of agents from large chemical collections and screened for ICD manifestations in a semi-automated manner, followed by in vitro validation experiments with alternative methods for ICD detection and additional cell lines.…”

“…More recently, we have used artificial intelligence to design algorithms that relate physicochemical descriptors of chemical agents with biological activity. These algorithms can predict the likelihood of distinct molecules to induce ICD, hence enabling the pre-selection of drugs with a high probability to operate as bona fide ICD inducers and hence reducing the cost of screening campaigns 149 , 206 .

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FLT3LG - a biomarker reflecting clinical responses to the immunogenic cell death inducer oxaliplatin

“…[1][2][3][4] In vitro screening for these hallmarks, in vivo vaccination experiments, as well as comparisons of anticancer drug effects in immunocompetent versus immunodeficient hosts, led to the identification of several pharmaceutical agents capable of inducing ICD. [5][6][7] Such ICD inducers include the platinum salt oxaliplatin (OXA), anthracyclines such as doxorubicin (DOX) or daunorubicin (DAU), as well as the alkylating agent cyclophosphamide. 4 Of note, certain chemotherapies that do not produce the whole spectrum of ICD hallmarks can complement each other to stimulate bona fide ICD.…”

Microbes, immunity and cancer in Capri: Another successful course of the EFIS‐EJI Ruggero Ceppellini Advanced School of Immunology founded by Serafino Zappacosta

“…Junior faculty member Oliver Kepp, from the Kroemer lab, gave a detailed account of how cellular stress ignites anti‐cancer responses and concentrated on the mechanisms and the clinical relevance of ICD. He also presented ways to couple a systems biology platform that screens compounds to ignite ICD with pre‐clinical mouse models of immunotherapy using those compounds .…”