(c) To assess ICD fingerprints, HT29 cells were treated with the indicated concentrations of KSPi


(c) To assess ICD fingerprints, HT29 cells were treated with the indicated concentrations of KSPi. of adenosine triphosphate (ATP) and high mobility group B1 protein (HMGB1), exposure of calreticulin on the cell surface as well as a transcriptional type-I interferon response. Further, in vivo experiments confirmed that treatment with TWEAKR-KSPi-ADCs activated immune responses via enhancing the infiltration of CD4+ and CD8+ T lymphocytes in tumors and the local production of interferon-, interleukin-2, and tumor necrosis factor-. In conclusion, the antineoplastic effects of TWEAKR-KSPi-ADCs can partly be attributed to its ICD-stimulatory properties. (( em i.p /em .) injections either with a single dose of 2.5 mg/kg TWEAKR-KSPi-ADC1 or with biweekly 5 mg/kg TWEAKR-KSPi-ADC2 for two weeks, respectively. The tumor area was measured by calipers three times per week followed by calculation of the tumor volume (Tvol?=?0.5 x length x width2). T/Cvol served as a parameter describing the ratio of mean tumor volumes of treated mice compared to the volume of vehicle control treated mice. However, when reducing the dose of the TWEAKR-KSPi-ADC1 to 2.5 mg/kg applied as a single dose, TWEAKR-KSPi-ADC1 and CTLA-4 blockade were equally efficacious in controlling tumor growth in immunocompetent mice (Figure 2c,d, Table 1), but at this low single dose of 2.5 mg/kg Rucaparib (Camsylate) TWEAKR-KSPi-ADC1 did not show any anti-tumor activity in immunodeficient mice (Figure 2e). In conclusion, it appears that the antineoplastic effect of TWEAKR-KSPi-ADC1 induces ICD in vivo and partly relies on the immune system. KSPi induces immunogenic cell death Since the active metabolite (Figure 1c) released from the TWEAKR-KSPi-ADC1 and -ADC2 (Figure 1a,B) is PKX1 not cell-permeable, we investigated a cell-permeable variant, the small molecule KSP inhibitor (Figure 1d) for induction of ICD. We took advantage of a predictive artificial intelligence tool to calculate ICD scores for each compound from the NCI60 collection and to compare their distribution with the one of the small molecule KSPi. In this approach, the small molecule KSPi had a predictive ICD score similar to the one of the prototype ICD inducer MTX (Figure 3) spurring our interest in further characterizing its potential in vitro. We thus validated the prediction by means of a phenotypic in vitro screening platform that combines the use of biosensors for the detection of ICD hallmarks with a fluorescence-based data Rucaparib (Camsylate) acquisition operating in a high-throughput format.19 The biosensors that we used included quinacrine, a small molecule that accesses the intracellular space and interacts with ATP in an acidic environment (mostly lysosomes) to emit a green fluorescence, meaning that the decrease of the intracellular fluorescent signal can be interpreted as a surrogate marker of ATP release (Figure 4). We also stably transfected cells with a green fluorescent protein (GFP) under the control of the type-I interferon-induced GTP-binding protein MX1, meaning that such cells constitute biosensors of type-I interferon responses (Figure 4). Alternatively, cancer cells were engineered to express an HMGB1-GFP fusion protein, allowing to monitor the release of HMGB1 from the nucleus (Figure 4). Finally, cells were equipped with a CALR-GFP fusion protein to follow the translocation of CALR from the ER to the cell periphery (Figure 4a,b). These biosensor experiments were calibrated in a way that mitoxantrone (MTX), an established ICD inducer, would induce all hallmarks of ICD (Figure 4). Of note, we found that the small molecule KSPi was as efficient as MTX in inducing the four ICD hallmarks in a time- and dose-dependent fashion in HT29 colorectal adenocarcinoma biosensor cells (Figure 4c, Supplemental Fig. S4) and two additional cell lines different in origin, namely, NCI-H929 myeloma and HCC70 mammary carcinoma (Supplemental Fig. S5C7). In conclusion, the small molecule KSPi can efficiently induce ICD. Open in a separate window Figure 3. Prediction of the potential of KSPi to induce immunogenic cell death. The immunogenic cell death Rucaparib (Camsylate) (ICD) scores were predicted for each compound from the NCI60 collection together with the KSPi used in this study by means of the ICDPred package (https://github.com/kroemerlab/ICDpred).19,51 The distribution of ICD scores was plotted (dashed lines indicate quantiles) showing that the KSPi and the prototype ICD inducer mitoxantrone (MTX) belong to the 20% and 10% highest scores, respectively. Figure 4. Biosensor cells for the immunogenic.