CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma
2021; Springer Nature; Volume: 13; Issue: 6 Linguagem: Inglês
10.15252/emmm.202012344
ISSN1757-4684
AutoresRosanna Mezzapelle, Francesco De Marchis, Chiara Passera, M. Di Leo, Francesca Brambilla, Federica Colombo, Maura Casalgrandi, Alessandro Preti, Samuel Zambrano, Patrizia Castellani, Riccardo Ertassi, Marco Silingardi, Francesca Caprioglio, Veronica Basso, Renzo Boldorini, Angelo Carretta, Francesca Sanvito, Ottavio Rena, Anna Rubartelli, Lina Sabatino, Anna Mondino, Massimo P. Crippa, Vittorio Colantuoni, Marco E. Bianchi,
Tópico(s)Immunotherapy and Immune Responses
ResumoArticle6 May 2021Open Access Source DataTransparent process CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma Rosanna Mezzapelle Rosanna Mezzapelle Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Francesco De Marchis Francesco De Marchis orcid.org/0000-0003-1275-5470 Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Chiara Passera Chiara Passera Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Manuela Leo Manuela Leo Department of Sciences and Technologies, University of Sannio, Benevento, Italy Search for more papers by this author Francesca Brambilla Francesca Brambilla Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Federica Colombo Federica Colombo Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milano, Italy Search for more papers by this author Maura Casalgrandi Maura Casalgrandi HMGBiotech S.r.l., Milan, Italy Search for more papers by this author Alessandro Preti Alessandro Preti HMGBiotech S.r.l., Milan, Italy Search for more papers by this author Samuel Zambrano Samuel Zambrano Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Patrizia Castellani Patrizia Castellani orcid.org/0000-0002-6326-5853 Cell Biology Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy Search for more papers by this author Riccardo Ertassi Riccardo Ertassi Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Marco Silingardi Marco Silingardi School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Francesca Caprioglio Francesca Caprioglio School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Veronica Basso Veronica Basso Division of Immunology, Transplantation and Infectious Diseases, Lymphocyte Activation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Renzo Boldorini Renzo Boldorini Department of Health Science, School of Medicine, University of Eastern Piedmont Amedeo Avogadro, Vercelli, Italy Pathology Unit, Maggiore della Carità Hospital, Novara, Italy Search for more papers by this author Angelo Carretta Angelo Carretta Department of Thoracic Surgery, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Francesca Sanvito Francesca Sanvito Department of Pathology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Ottavio Rena Ottavio Rena Unit of Thoracic Surgery, Maggiore della Carità Hospital, Novara, Italy Search for more papers by this author Anna Rubartelli Anna Rubartelli Cell Biology Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy Search for more papers by this author Lina Sabatino Lina Sabatino Department of Sciences and Technologies, University of Sannio, Benevento, Italy Search for more papers by this author Anna Mondino Anna Mondino Division of Immunology, Transplantation and Infectious Diseases, Lymphocyte Activation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Massimo P Crippa Massimo P Crippa Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Vittorio Colantuoni Corresponding Author Vittorio Colantuoni [email protected] Department of Sciences and Technologies, University of Sannio, Benevento, Italy Search for more papers by this author Marco E Bianchi Corresponding Author Marco E Bianchi [email protected] Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Rosanna Mezzapelle Rosanna Mezzapelle Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Francesco De Marchis Francesco De Marchis orcid.org/0000-0003-1275-5470 Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Chiara Passera Chiara Passera Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Manuela Leo Manuela Leo Department of Sciences and Technologies, University of Sannio, Benevento, Italy Search for more papers by this author Francesca Brambilla Francesca Brambilla Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Federica Colombo Federica Colombo Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milano, Italy Search for more papers by this author Maura Casalgrandi Maura Casalgrandi HMGBiotech S.r.l., Milan, Italy Search for more papers by this author Alessandro Preti Alessandro Preti HMGBiotech S.r.l., Milan, Italy Search for more papers by this author Samuel Zambrano Samuel Zambrano Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Patrizia Castellani Patrizia Castellani orcid.org/0000-0002-6326-5853 Cell Biology Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy Search for more papers by this author Riccardo Ertassi Riccardo Ertassi Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Marco Silingardi Marco Silingardi School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Francesca Caprioglio Francesca Caprioglio School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Veronica Basso Veronica Basso Division of Immunology, Transplantation and Infectious Diseases, Lymphocyte Activation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Renzo Boldorini Renzo Boldorini Department of Health Science, School of Medicine, University of Eastern Piedmont Amedeo Avogadro, Vercelli, Italy Pathology Unit, Maggiore della Carità Hospital, Novara, Italy Search for more papers by this author Angelo Carretta Angelo Carretta Department of Thoracic Surgery, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Francesca Sanvito Francesca Sanvito Department of Pathology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Ottavio Rena Ottavio Rena Unit of Thoracic Surgery, Maggiore della Carità Hospital, Novara, Italy Search for more papers by this author Anna Rubartelli Anna Rubartelli Cell Biology Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy Search for more papers by this author Lina Sabatino Lina Sabatino Department of Sciences and Technologies, University of Sannio, Benevento, Italy Search for more papers by this author Anna Mondino Anna Mondino Division of Immunology, Transplantation and Infectious Diseases, Lymphocyte Activation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Massimo P Crippa Massimo P Crippa Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Vittorio Colantuoni Corresponding Author Vittorio Colantuoni [email protected] Department of Sciences and Technologies, University of Sannio, Benevento, Italy Search for more papers by this author Marco E Bianchi Corresponding Author Marco E Bianchi [email protected] Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy School of Medicine, Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Author Information Rosanna Mezzapelle1,2, Francesco De Marchis1, Chiara Passera1, Manuela Leo3, Francesca Brambilla1, Federica Colombo1,4, Maura Casalgrandi5, Alessandro Preti5, Samuel Zambrano1,2, Patrizia Castellani6, Riccardo Ertassi1, Marco Silingardi2, Francesca Caprioglio2, Veronica Basso7, Renzo Boldorini8,9, Angelo Carretta10, Francesca Sanvito11, Ottavio Rena12, Anna Rubartelli6, Lina Sabatino3, Anna Mondino7, Massimo P Crippa1, Vittorio Colantuoni *,3 and Marco E Bianchi *,1,2 1Chromatin Dynamics Unit, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy 2School of Medicine, Vita-Salute San Raffaele University, Milan, Italy 3Department of Sciences and Technologies, University of Sannio, Benevento, Italy 4Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milano, Italy 5HMGBiotech S.r.l., Milan, Italy 6Cell Biology Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy 7Division of Immunology, Transplantation and Infectious Diseases, Lymphocyte Activation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy 8Department of Health Science, School of Medicine, University of Eastern Piedmont Amedeo Avogadro, Vercelli, Italy 9Pathology Unit, Maggiore della Carità Hospital, Novara, Italy 10Department of Thoracic Surgery, IRCCS San Raffaele Scientific Institute, Milan, Italy 11Department of Pathology, IRCCS San Raffaele Scientific Institute, Milan, Italy 12Unit of Thoracic Surgery, Maggiore della Carità Hospital, Novara, Italy *Corresponding author. Tel: +39 0824 305102; E-mail: [email protected] *Corresponding author. Tel: +39 0226 434762; E-mail: [email protected] EMBO Mol Med (2021)13:e12344https://doi.org/10.15252/emmm.202012344 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Boosting antitumor immunity has emerged as a powerful strategy in cancer treatment. While releasing T-cell brakes has received most attention, tumor recognition by T cells is a pre-requisite. Radiotherapy and certain cytotoxic drugs induce the release of damage-associated molecular patterns, which promote tumor antigen cross-presentation and T-cell priming. Antibodies against the “do not eat me” signal CD47 cause macrophage phagocytosis of live tumor cells and drive the emergence of antitumor T cells. Here we show that CXCR4 activation, so far associated only with tumor progression and metastasis, also flags tumor cells to immune recognition. Both CXCL12, the natural CXCR4 ligand, and BoxA, a fragment of HMGB1, promote the release of DAMPs and the internalization of CD47, leading to protective antitumor immunity. We designate as Immunogenic Surrender the process by which CXCR4 turns in tumor cells to macrophages, thereby subjecting a rapidly growing tissue to immunological scrutiny. Importantly, while CXCL12 promotes tumor cell proliferation, BoxA reduces it, and might be exploited for the treatment of malignant mesothelioma and a variety of other tumors. Synopsis Induction of antitumor immunity is a successful strategy in cancer treatment. This study reports that BoxA, a fragment of the alarmin HMGB1, induces tumor remission and antitumor immunity in mouse models of mesothelioma and colon carcinoma. Both BoxA and the chemokine CXCL12 bind the G-Protein Coupled Receptor CXCR4. CXCR4 and CD47 are in contact on the surface of tumor cells and co-internalize upon CXCR4 engagement by either BoxA or CXCL12. Both CXCL12 and BoxA induce the phagocytosis of tumor cells by macrophages. BoxA inhibits tumor cell growth and induces antitumor immunological memory in syngeneic mouse models of mesothelioma or colon carcinoma. CXCL12 is suggested to mediate a similar response (Immunogenic Surrender) in a fraction of untreated tumor-bearing mice. The paper explained Problem Malignant mesothelioma (MM) is a tumor arising from asbestos-induced chronic inflammation and for which few therapeutic options are available. High Mobility Group Box 1 (HMGB1) protein favors the onset and progression of MM. We tested the therapeutic potential of BoxA, a fragment of HMGB1 that competes with the intact protein, in an immune-competent mouse model of MM. Results We find that BoxA induces MM remission and antitumor immunization in a large fraction of mice. The binding of BoxA to the CXCR4 receptor induces DAMPs release and CD47 internalization, leading to tumor cell phagocytosis by macrophages. CXCL12, the natural ligand of CXCR4, also promotes CD47 internalization. Impact Our study indicates that the CXCL12/CXCR4 axis, which is known to promote cancer progression, also promotes a counterbalancing antitumor response. BoxA is non-toxic and, contrary to CXCL12, inhibits tumor cell growth. Thus, BoxA, by shifting the balance from tumor growth to antitumor immunization, might hold promise as first-in-class antitumor drug that should be synergic with checkpoint inhibitors. Furthermore, synthetic ligands that act like BoxA may be as effective as anti-CD47 antibodies, which are in advanced clinical development. Introduction Chronic inflammation and the presence of an unfavorable inflammatory microenvironment can promote tumor development. A common example is colon carcinoma (Terzić et al, 2010), but as representative is malignant mesothelioma (MM), a tumor that is associated with asbestos exposure and comprises a large inflammatory component, in particular macrophages (Lievense et al, 2013). We previously showed that both MM cells and macrophages secrete High Mobility Group Box 1 protein (HMGB1) (Yang et al, 2010; Jube et al, 2012), an alarmin that alerts the innate and adaptive immune systems to tissue damage and cell stress (Bianchi et al, 2017). HMGB1 plays a central role in tissue regeneration, in part by recruiting monocytes/macrophages via the CXCR4 receptor and directing them toward a tissue-healing phenotype (Tirone et al, 2018). In MM, secreted HMGB1 sustains chronic inflammation initially caused by asbestos and supports disease progression (Jube et al, 2012; Xue et al, 2020). HMGB1 has several receptors, among which TLR4, RAGE, and CXCR4 are the most well-known (Bianchi et al, 2017). BoxA is a fragment of HMGB1 that corresponds to its first HMG-box domain and competes with HMGB1 for binding to the RAGE and TLR4 without activating them (Venereau et al, 2016; He et al, 2018). We previously reported that targeting HMGB1 with monoclonal antibodies or BoxA extends the survival of mice xenografted with human MM cells by interfering with tumor cell proliferation (Yang et al, 2015). However, extracellular HMGB1 also primes antigen recognition (Rovere-Querini et al, 2004) and is involved in immunogenic cell death (ICD). ICD is induced by certain chemotherapeutics or radiotherapy and increases the processing of apoptotic tumor cells by dendritic cells (DCs), enhances their immunogenicity, and elicits an efficient antitumor immune response and immunological memory (Galluzzi et al, 2020) The mechanism of ICD involves the apoptosis of tumor cells, preceded by endoplasmic reticulum (ER) stress, with concomitant induction of the unfolded protein response (UPR) and the release of HMGB1, ATP, and calreticulin (Kroemer et al, 2012). Calreticulin is an abundant ER-resident protein that becomes an “eat me” signal once relocated to the cell surface (ecto-calreticulin). To test whether targeting HMGB1 is beneficial or detrimental in immunocompetent tumor-bearing hosts, we set up a syngeneic model of MM, where mouse AB1 malignant mesothelioma cells are grafted into the peritoneum of syngeneic BALB/c mice (Mezzapelle et al, 2016). Surprisingly, we found that BoxA, besides being antiproliferative, also promotes protective antitumor immunity responsible for MM rejection and long-term survival in a large fraction of mice. Exploration of the mode of action of BoxA revealed that it acts via CXCR4. CXCR4 is a G-protein coupled receptor that induces cell migration upon binding its main ligand CXCL12 (also known as SDF-1) (Teicher & Fricker, 2010; Bianchi & Mezzapelle, 2020). CXCR4 is also involved in metastatisation, and in many types of tumors upregulation of CXCR4 and of its ligand CXCL12 are predictive of short disease-free survival (Guo et al, 2016). Robust upregulation of CXCR4 was reported in human mesothelioma cell lines and in mesothelioma tissues (Li et al, 2011). However, in MM cells engagement of CXCR4 by BoxA does not promote cell growth but rather induces the surface exposure of calreticulin and the depletion of surface CD47, tilting the balance of “eat me” and “don’t eat me” signals, and promoting tumor cell phagocytosis by macrophages. CD47 is a ubiquitous transmembrane protein that prevents the phagocytosis of functionally fit cells by interacting with its ligand SIRP1α (signal regulatory protein 1α) on the surface of macrophages and DCs (Barclay & van den Berg, 2014). Lack of CD47 on erythrocytes, platelets, and lymphohematopoietic cells results in rapid clearance of these cells by macrophages (Blazar et al, 2001). CD47 is expressed at increased level on the cell surface by a variety of malignant cells (Willingham et al, 2012); its blockade with monoclonal antibodies allows the efficient phagocytosis of cancer cells and leads to tumor rejection and development of antitumor immunity (Liu et al, 2015). CD47 blockade has remarkable therapeutic efficacy in various preclinical models of bladder, colon and breast cancer, glioblastoma, lymphoma, and acute lymphocytic leukemia (Jaiswal et al, 2009; Willingham et al, 2012; Liu et al, 2015). The published studies involve the masking of CD47 by antibodies that prevent its interaction with SIRP1α; whether and how CD47 exposure is modulated in response to the microenvironment is still unknown. Here, we show that CXCR4 engagement promotes the internalization of CD47 and the downstream antitumor responses, both when triggered by BoxA or CXCL12. Thus, we argue that the CXCL12/CXCR4 axis activates immunosurveillance via a mechanism (which we name Immunogenic Surrender) that allows tumor identification by innate cells and tumor-specific T-cell priming. Results BoxA promotes tumor rejection and the development of protective antitumor immune memory HMGB1 promotes human MM cell survival and proliferation via RAGE (Jube et al, 2012), whereas BoxA, its N-terminal fragment, acts as an HMGB1 competitor and antagonist on RAGE and TLR4 receptors (Venereau et al, 2016). Accordingly, BoxA was found to reduce tumor growth and extend mice survival in a model where human MM cells were injected into immunodeficient mice (Jube et al, 2012; Yang et al, 2015). However, HMGB1 plays a key role in inducing ICD (Kroemer et al, 2012), and therefore, targeting HMGB1 might reduce antitumor immune responses. To investigate the antitumor potential of BoxA in immune-competent mice and possible underlying mechanisms, we exploited the syngeneic mouse model of MM we previously developed, where mouse AB1-B/c MM cells are engrafted in the peritoneum of BALB/c mice (Mezzapelle et al, 2016). Inoculation of 7 × 104 MM cells produced MM tumors (Fig 1A) that were highly infiltrated by inflammatory cells, mostly represented by CD206+ CD86− macrophages, and few CD3+ T and B cells (Fig EV1A and B). HMGB1 was highly expressed both in the nucleus and in the cytosol of tumor cells. This pattern is very similar to that of human mesothelioma (Fig EV1C). Figure 1. BoxA increases survival and induces immunization in a syngeneic mouse model of mesothelioma A. Scheme of the experiment. BALB/c mice were inoculated i.p. with 7 × 104 MM cells and treated with either 800 µg BoxA (red bars) or PBS (blue bars) three times a week, 10 times in total. Yellow arrows represent BLI imaging. B. Treatment with BoxA at increasing doses reduced the number of mice with detectable tumor lesions in a statistically significant dose-dependent manner (Spearman correlation: P = 0.04). In this experiment, no BLI measurement was taken. C. Forty mice were inoculated with MM cells and treated or not with BoxA. Tumor growth was detected via BLI. Lines that do not reach day 34 correspond to mice that were sacrificed for ethical reasons. D. Kaplan–Meier survival curves. Statistics: log-rank Gehan–Breslow–Wilcoxon test, P < 0.0001, n = 20 per group. E. Tumor growth after MM re-challenge detected by BLI. F–G. Flow cytometry analysis of luciferase-specific T cells. In (F), representative dot plots depict intracellular IFNγ levels in gated CD8+ CD44high T cells. In (G), differences in the percentage of IFNγ producing CD8+ CD44high cells, pulsed relative to unpulsed. Statistics: Kruskal–Wallis test; each dot represents a mouse (n = 3–7 per group); bars represent mean ± SD. Source data are available online for this figure. Source Data for Figure 1 [emmm202012344-sup-0004-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Immunohistochemical survey of tumor microenvironment in mesothelioma Representative IHC staining for F4/80, CD45R, CD3, and HMGB1 in two mouse mesotheliomas. Nuclei were counterstained with hematoxylin. Scale bar 20 µm. Representative immunofluorescence (IF) staining for F4/80 (red), CD86 (green, in the upper panel), and CD206 (green, in the lower panel) of mouse MM lesions. Scale bar 50 µm. Representative IHC staining for CD68, CD206, CD163, CD20, CD3, and HMGB1 in human sarcomatoid and epithelioid mesotheliomas. Scale bar 50 µm. Download figure Download PowerPoint In a first small-scale experiment, we inoculated AB1-B/c mouse MM cells in the peritoneum of 12 BALB/c mice, and 3 days later, we started treatment with 0, 200, 400, and 800 µg BoxA, three times a week. After 22 days, all control mice had developed tumor lesions, while mice treated with 800 µg BoxA had no discernible lesions, and mice treated with smaller doses of BoxA had an intermediate incidence (Fig 1B). Thus, BoxA was not toxic at the highest dose (800 µg per injection) and showed antitumor effects also in immunocompetent mice. To follow tumor development in longitudinal analysis by BioLuminescence Imaging (BLI), we adopted AB1 cells expressing luciferase (AB1-B/c-LUC, henceforth called MM cells). Mice received i.p. delivery of BoxA or PBS (control) 2 days after MM injection (Fig 1A). At day 6, the high level of abdominal BLI signal (Fig 1C) indicated the engraftment of MM cells in both control and BoxA-treated mice. In the following weeks, most control mice experienced an increase of the BLI signal and had to be sacrificed. Notably, 4 control mice (20%) showed a tenfold decrease of the BLI signal relative to the first measurement, but then experienced remission and survived beyond the end of the 3-week treatment (Fig 1C and D). In contrast, 18 BoxA-treated mice showed a decrease of the BLI signal after day 6, in some cases to 10-fold below the initial measurement, and 15 (75%) survived after the end of the treatment. At day 75, the difference in survival curves between control mice and BoxA-treated mice was highly significant (P < 0.0001) (Fig 1D). We sacrificed two of the surviving mice per group (control and BoxA treated) and we could not identify any tumor mass, either in the abdomen or elsewhere; this difference in tumor rejection was highly significant (4/20 versus 15/20, P = 0.0012 Fisher’s test). These results indicate that BoxA treatment can extend the survival of model MM mice, but most of all increases the fraction of mice that reject the tumor. Notably, the efficacy of BoxA appears strikingly higher in immunocompetent mice compared to immunodeficient ones (Yang et al, 2015), suggesting that the immune system is an active player in the activity of BoxA. To test whether surviving mice had developed immunological memory against the tumor, we re-challenged them with MM cells. All mice showed a high level of bioluminescence soon after the re-challenge, but only background levels 7 days later; all of them survived for several weeks without signs of disease (Fig 1E). We then repeated the experiment described in Fig 1B and tested for the presence of tumor-specific CD8+ T memory cells, exploiting luciferase as surrogate tumor-associated antigen (Limberis et al, 2009). We recovered splenocytes from 4 groups of mice: (i) naïve (not injected with MM cells, not treated), (ii) injected with MM cells and surviving after being treated with control (PBS), (iii) injected with MM cells and sacrificed because of tumor progression despite being treated with BoxA (BoxA not cured), and (iv) injected with MM cells and surviving after being treated with BoxA (BoxA cured). The splenocytes were cultured for 5 days in the presence of the luciferase peptide GFQSMYTFV to expand LUC-specific T cells and then stimulated (pulsed) or not (unpulsed) for 4 h with the Luc peptide. Splenocytes from BoxA-cured mice contained a significantly higher percentage of CD8+CD44high IFNγ-producing T cells upon peptide stimulation than splenocytes from mice of the other treatment groups (Fig 1F and G; P < 0.005, Kruskal–Wallis test). This experiment indicates that BoxA promotes T-cell responses to a surrogate tumor-associated antigen. In spontaneously surviving mice (not treated with BoxA), the absence of detectable populations of LUC-specific T cells may reflect sub-optimal priming and a relative paucity of antitumor T-cell clones. We also tested the requirement for T cells in BoxA-dependent antitumor responses. We depleted mice of CD8+ T cells (Appendix Fig S1) prior to the inoculation of MM cells, and then we treated them with either BoxA or PBS (Fig 2A). All CD8-depleted mice developed tumors regardless of treatment (BoxA or PBS) and were sacrificed after 2 weeks. In contrast, some of the non-depleted control mice survived longer than their control counterpart, and this was further promoted by BoxA (Fig 2B and C). Figure 2. BoxA exerts its therapeutic effect through CD8+ T cells Scheme of the experiment. BALB/c mice were depleted or not of CD8+ T cells 3 days before the injection of 7 × 104 MM cells and subsequently treated with either 800 µg BoxA (n = 9) or PBS (n = 7) three times a week, for 10 times in total. Every week mice were surveyed by BLI. Representation of the BLI of the four groups of mice. Left panel: CD8+ T-cell depleted mice treated with BoxA (n = 9) or PBS (n = 7). Right panel: non-depleted mice treated with BoxA (n = 11) or PBS (n = 7). Kaplan–Meier survival curves of the 4 groups of mice shown in panel (B). Statistics: Gehan–Breslow–Wilcoxon test. Source data are available online for this figure. Source Data for Figure 2 [emmm202012344-sup-0005-SDataFig2.xlsx] Download figure Download PowerPoint Overall our results show that transplantation of MM cells evokes a spontaneous protective immune response, which can lead to tumor rejection and immunological memory in a small fraction of mice. BoxA boosts immune-mediated recognition, increases the number of T cells that recognize tumor-associated antigens, and increases the fraction of mice that develop long-term antitumor immunity. BoxA induces the relocation of calreticulin without causing cell death We had expected that BoxA might interfere with ICD, but the results reported in the previous section showed that BoxA favors antitumor immune responses and immunization. We then investigated whether BoxA might instead promote ICD. BoxA induced an increase in the surface exposure of calreticulin to an extent comparable to the well-known ICD inducer mitoxantrone (MTX) (Figs 3A and EV2A). Tumor masses explanted from mice treated with BoxA displayed calreticulin on the surface of cells, contrary to tumors from untreated mice (Fig 3B). BoxA also induced the release of HMGB1 (Fig 3C), although less efficiently than MTX, and the phosphorylation of eIF2α (Fig 3D), which is pathognomonic for ICD (Bezu et al, 2018). However, we detected a transient and dose-dependent activation of each of the three branches of the UPR (Fig EV2-EV6), whereas MTX activated only the PERK-eIF2α arm (Fig 3D), in line with data from the literature (Panaretakis et al, 2009). Figure 3. BoxA induces the release of damage-associated molecular pattern molecules (DAMPs) but not apoptosis in MM cells Surface calreticulin on MM cells was evalua
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