Calreticulin exposure correlates with robust adaptive antitumor immunity and favorable prognosis in ovarian carcinoma patients
Adjuvanticity, which is the ability of neoplastic cells to deliver danger signals, is critical for the host immune system to mount spontaneous and therapy-driven anticancer immune responses. One of such signals, i.e., the exposure of calreticulin (CALR) on the membrane of malignant cells experiencing endoplasmic reticulum (ER) stress, is well known for its role in the activation of immune responses to dying cancer cells. However, the potential impact of CALR on the immune contexture of primary and metastatic high-grade serous carcinomas (HGSCs) and its prognostic value for patients with HGSC remains unclear.
We harnessed a retrospective cohort of primary (no = 152) and metastatic (no = 74) tumor samples from HGSC patients to investigate the CALR expression in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 302 HGSC samples was used as a confirmatory approach.
We demonstrate that CALR exposure on the surface of primary and metastatic HGSC cells is driven by a chemotherapy-independent ER stress response and culminates with the establishment of a local immune contexture characterized by TH1 polarization and cytotoxic activity that enables superior clinical benefits.
Our data indicate that CALR levels in primary and metastatic HGSC samples have robust prognostic value linked to the activation of clinically-relevant innate and adaptive anticancer immune responses.
KeywordsB cells Cancer immunotherapy CD20 DC-LAMP Dendritic cells Immunogenic cell death
It is now accepted that tumors form, progress and respond to therapy in the context of an intimate, bidirectional interaction with the immune system [1, 2]. In this context, malignant cells progressively escape immunosurveillance by losing their antigenicity, i.e., the exposure on the cell surface of antigens not covered by central thymic tolerance [3, 4] and adjuvanticity, i.e., the emission of immunostimulatory signals through molecules commonly known as damage-associated molecular patterns (DAMPs) [5, 6]. In physiological conditions, DAMPs are sequestered in the intracellular microenvironment, where they cannot be detected by the host immune system [5, 6]. However, cells experiencing sub-lethal or lethal stress conditions passively release, actively secrete, or expose on the outer leaflet of the plasma membrane, several DAMPs, hence enabling the latter to mediate a variety of immunomodulatory functions [7, 8, 9].
Endoplasmic reticulum (ER) chaperones including calreticulin (CALR) and various heat-shock proteins (HSPs) are well known for their key role as pro-phagocytic DAMPs in the successful activation of anticancer immunity by malignant cells undergoing immunogenic cell death [5, 6]. In line with this notion, high expression levels of CALR and/or CALR exposure on the membrane of cancer cells have been linked with superior disease outcome in patients with colorectal carcinoma (CRC) , non-small cell lung carcinoma (NSCLC) [11, 12], acute myeloid leukemia (AML) , and ovarian cancer  generally in association with improved anticancer immunity. Conversely, the impact of CALR levels on the composition and functional orientation of the HGSC microenvironment remain unclear.
Here, we investigated the impact of CALR levels on disease outcome in a retrospective cohort of 152 patients with resectable high-grade serous carcinoma (HGSC) who did not receive neoadjuvant chemotherapy. Our data suggest that increased CALR levels in both primary and metastatic tumor tissues are associated with superior disease outcome linked to the establishment of a tumor microenvironment (TME) exhibiting TH1 polarization and activation of immune effectors.
Materials and methods
Main clinicopathological features of Study Group 1
Study Group 1
(n = 152)
Mean age ± SEM
65 ± 0.81
Vital status of patients
Tumor specimens from Study Group 1 and Study Group 2 were fixed in neutral buffered 10% formalin solution and embedded in paraffin as per standard procedures. Immunostaining with antibodies specific for lysosomal associated membrane protein 3 (LAMP3; best known as DC-LAMP), CD8, CD20, CALR and natural cytotoxicity triggering receptor 1 (NCR1; best known as NKp46) (Additional file 1: Table S3) was performed according to conventional protocols. Briefly, tissue sections were deparaffinized and rehydrated descending alcohol series (100, 96, 70, and 50%), followed by antigen retrieval with Target Retrieval Solution (Leica) in EDTA pH 8.0 (for DC-LAMP/CD20, CD8, NKp46) or in citrate buffer at pH 6.0 (for CALR), in preheated water bath (97 °C, 30 min). Sections were allowed to cool down to RT for 30 min, and endogenous peroxidase was blocked with 3% H2O2 for 15 min. For co-staining, endogenous alkaline phosphatase was blocked by levamisole (Vector). Sections were then treated with protein block (DAKO) for 15 min and incubated with primary antibodies, followed by the revelation of enzymatic activity. Images were acquired using a Leica Aperio AT2 scanner (Leica).
CALR expression in the tumor microenvironment was quantified as a function of CALR+ positive tumor cells, as published previously . Scores were calculated on 10 different fields visually inspected at 20x magnification under a light microscope (DM2000LED; Leica), and classified into (1) score 1, < 10% CALR+ cells; (2) score 2, 10–25% CALR+ cells, (3) score 3, 26–50% CALR+ cells; (4) score 4, 51–75% CALR+ cells; and (5) score 5, > 75% positive cells (Additional file 1: Figure S2.). Quantification was done performed by two independent observers (LK, JF) and reviewed by two expert pathologists (JL, PS). DC-LAMP+, CD8+, CD20+ and NKp46+ cells were quantified in the tumor stroma and tumor nests of the whole tumor sections with Calopix (Tribvn). Data are reported as absolute number of positive cells/mm2 (for DC-LAMP+, CD8+ and NKp46+ cells) or cell surface/total tumor section surface (for CD20+ cells), as previously described [16, 17, 18, 19]. Immunostaining and quantifications were reviewed by at least three independent observers (IT, LK, JF, PS, JL) and two expert pathologists (JL, PS).
As previously described, fresh ovarian tumor specimens were minced with scissors, digested in PBS containing 1 mg/ml of Collagenase D (Roche) and 0.2 mg/ml DNase I at 37 °C for 30 min mechanically dissociated using the gentleMACS dissociator (Miltenyi Biotec) and passed through a 70 μm nylon cell strainer (BD Biosciences) . To determine the ecto-CALR exposure, mononuclear cells were stained with primary antibodies against CD45, cytokeratin, human epithelial antigen, CD227 to distinguish the population of leukocytes and malignant cells, and antibodies against CALR or isotype control (Additional file 1: Table S4) for 20 min at 4 °C in the dark, following by washing and acquisition on a Fortessa flow cytometer (BD Bioscience). Flow cytometry data were analyzed with the FlowJo software (TreeStar). Gating strategy is depicted in Additional file 1: Figure S3.
Degranulation and IFN-γ production after in vitro stimulation
Mononuclear cells isolated from fresh tumor specimens were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) + 1 μg/ml of ionomycin for 1 h followed by 3 h incubation with brefeldin A (BioLegend). Unstimulated cells were used as a control. The cells were then washed in PBS, stained with anti-CD3 Alexa Fluor 700 (EXBIO), anti-CD4 ECD (Beckman Coulter) and anti-CD8 HV500 (BD Biosciences), fixed using fixation/permeabilization buffer (eBioscience), permeabilized with permeabilization buffer (eBioscience) and intracellularly stained with an anti-IFN-γ PE-Cy7 (eBioscience), anti-granzyme B Brilliant Violet 421 (BD Biosciences) (Additional file 1: Table S4). The percentage of CD3+CD8+ T cells producing IFN-γ and degranulating upon PMA/ionomycin stimulation were determined by flow cytometry. The data were analyzed with the FlowJo software package (Tree Star, Inc.). Gating strategy is depicted in Additional file 1: Figure S4.
TCGA data analysis
Patients with HGSC (n = 302) were identified in The Cancer Genome Atlas (TCGA) public database (https://cancergenome.nih.gov/). Differentially expressed genes (DEGs) between the CALRHi and CALRLo groups were determined using the LIMMA-R package . Hierarchical clustering analysis was conducted using the ComplexHeatmap package, based on the Euclidean distance and complete clustering method . Immune analyses were performed using ClueGo . The MCP-counter R package was used to estimate the abundance of tissue-infiltrating immune cell populations (Additional file 1: Table S5) .
Survival analysis was performed using the R package survival analysis. The overall prognostic value of continuous variables was assessed (1) by Wald tests for univariate COX regression models, (2) by log-rank tests using median-based cutoffs. The prognostic value of CALR and immune density was assessed by multivariate Cox regression. Student’s t tests, Wilcoxon tests and Mann-Whitney tests were used to assess statistical significance, p values are reported (considered not significant when > 0.05).
Prognostic impact of CALR expression in TME of primary and metastatic HGSC
Univariate Cox proportional hazard analysis
HR (95% Cl)
HR (95% Cl)
Multivariate Cox proportional hazard analysis
HR (95% Cl)
HR (95% Cl)
CALR levels in HGSC correlate with signs of an ongoing ER stress response
High CALR levels are associated with a TH1-polarized, cytotoxic CD8+ T-cell response
CALR expression is associated with HGSC infiltration by activated DCs and B cells
CALR levels are associated with HGSC infiltration by IFN-γ producing CD8+ T cells
As we observed a positive correlation between CALR levels and tumor infiltration by diverse immune cell subsets, we next evaluated the global immunological profile of the TME of CALRLo versus CALRHi PT samples from Study Group 1 by IHC. This approach identified 4 different clusters of patients corresponding to high versus low CALR expression in the context of elevated versus reduced tumor infiltration by DC-LAMP+ mature DCs, CD20+ B cells and CD8+ T cells (ImmuneHi and ImmuneLo, respectively) (Fig. 5h). Importantly, CALR status improved the prognostic assessment on RFS and OS amongst both ImmuneHi (RFS: p = 0.01; OS: p = 0.01) and ImmuneLo (RFS: p = 0.008; OS: p = 0.02) patient subgroups (Fig. 5i). Altogether, our findings document a robust independent prognostic value for CALR levels of chemotherapy-naïve patients with HGSC, linked to the impact of CALR on the establishment of a TH1-polarized TME that supports anticancer immunity.
Despite recent developments in diagnostic and treatment modalities leading to an improvement in the short-term survival of patients with ovarian cancer, most of patients are diagnosed at advanced stage of the disease with metastatic spreading, due to the non-specific symptoms and the absence of effective screening methods . Therefore, there is an urgent need for new diagnostic, including prognostic and predictive biomarkers and therapeutic tools for a clinical management of cancer patients, which still represents the principal cause of mortality from gynecologic malignancies. Accumulating preclinical and clinical evidence indicates that DAMPs and DAMP-associated processes impact disease outcome in patients with various malignancies . In particular, the prognostic relevance of CALR expression levels or exposure on the membrane of cancer cells has been investigated by us and others in the context of multiple malignancies [10, 11, 12, 13, 32, 33, 34]. Nevertheless, the influence of CALR levels on the composition and functional orientation of the immune infiltrate of HGSCs and their link with disease outcome in chemotherapy-naïve patients remain have not been elucidated until now.
As documented in numerous in vitro and in vivo models, ecto-CALR serves as a signal to facilitate the engulfment of tumor cells by DCs, which leads to tumor antigen presentation and stimulation tumor-specific cytotoxic T lymphocytes responses [35, 36]. Here, we analyzed 3 different cohorts of primary and metastatic samples from patients with HGSCs who did not receive chemotherapy prior to tumor resection. By combining IHC and biomolecular analyses, we demonstrated that a high CALR expression is strongly associated with higher density of both mature DC-LAMP+ DCs and CD20+ B cells resulting in TH1-polarized immune contexture that acquired effector functions. These findings recapitulate previous findings by us and others demonstrating that CALR exposure by neoplastic cells is associated with increased tumor infiltration by myeloid cells and effector memory CD8+ T cells in patients with NSCLC , increased frequency of T cells in TME of colorectal carcinoma  and increased proportion of LAA-specific CD4+ and CD8+ T cells in patients with AML . Moreover, here we observed correlation between high CALR expression in the TME and higher cytotoxic functions of effector tumor infiltrating CD8+ T cells and NK cells, although the number of later population was not significantly increased in CALRHi patients, suggesting the impact of CARL exposure on enhanced NK cell cytotoxic and secretory functions. These results are in line with our recent findings demonstrating that spontaneous CALR exposure on malignant blasts supports innate anticancer immunity by NK cells via and indirect mechanism relying on myeloid CD11c+CD14+ cells resulting in overall superior survival of AML patients [37, 38]. Altogether, we demonstrated that high CALR levels bear independent positive prognostic value and hence can be harnessed to improve patient stratification based on previously identified factors including DC-LAMP+ DC, CD20+ B cell and CD8+ T cell infiltration. These findings extend previous data by us and others on the improved immunological functions linked to increased CALR levels in the context of AML , NSCLC  and CRC .
We also demonstrate that CALR is expressed by HGSC cells independent of standard-of-care chemotherapy, possibly reflecting malignant transformation itself  and/or the limited immunogenicity of carboplatin-based chemotherapy . Accordingly, we identified a robust correlation between CALR expression and 3 distinct genes involved in ER stress responses in two independent HGSC patient cohorts. Similar observations have been made by us and others in the context of AML [13, 41] and NSCLC . Interestingly, we also identified a significant decreased in CALR expression in samples from advanced stages of disease, which is in line with the notion that progressing tumors tend to lose both antigenicity and adjuvanticity [3, 5, 42].
In conclusion, CALR stand out as a robust prognostic biomarker for chemotherapy-naïve patients with HGSC. It can be speculated that CALRLo patients would benefit from neoadjuvant or adjuvant chemotherapeutic regimens that are known to drive robust ER stress responses in the context of ICD, such as oxaliplatin, doxorubicin and other anthracyclines . As ovarian cancer still represents one of the top 5 leading causes of cancer-related death amongst women in the US (source https://www.cdc.gov/cancer/uscs/index.htm), clinical trials specifically addressing this possibility are urgently awaited.
LG provides remunerated consulting to OmniSEQ (Buffalo, NY, USA), Astra Zeneca (Gaithersburg, MD, USA), VL47 (New York, NY, USA) and the Luke Heller TECPR2 Foundation (Boston, MA, USA), and he is member of the Scientific Advisory Committee of OmniSEQ (Buffalo, NY, USA). Dr. Kroemer reports grants and personal fees from Bayer Healthcare and grants from Genentech, Glaxo Smyth Kline, Lytix Pharma, PharmaMar, Sotio and Vasculox. He is member of the executive board of Bristol Myers Squibb Foundation France, as well as scientific co-founder of everImmune and Samsara therapeutics, outside of the submitted work.
Concept and design: PS, JL, AR, LG, RS, JF; development of the methodology: LK, MH, IT; acquisition of the data: LK, MH, IT, LB, JF; analysis and interpretation of the data: LK, MH, IT, PS, JL, IP, SV, MH, TB, LR, JP, JK; preparation, review, and/or revision of the manuscript and Figs: LK, MH, IC, LG, GK, RS, JF; study supervision: LR, RS, JF. All authors read and approved the final manuscript.
This study was supported by Sotio, Prague, Czech Republic; by the program PROGRES Q40/11 and PROGRES 28 (Oncology), by the project BBMRI-CZ LM2015089 and by the European Regional Development Fund-Project BBMRI-CZ.: Biobank network – a versatile platform for research on the etiopathogenesis of diseases, No: EF16 013/0001674. LG is supported by a Breakthrough Level 2 grant from the US Department of Defense (DoD), Breast Cancer Research Program (BRCP) (#BC180476P1), by a startup grant from the Dept. of Radiation Oncology at Weill Cornell Medicine (New York, US), by industrial collaborations with Lytix (Oslo, Norway) and Phosplatin (New York, US), and by donations from Phosplatin (New York, US), the Luke Heller TECPR2 Foundation (Boston, US) and Sotio a.s. (Prague, Czech Republic).
Ethics approval and consent to participate
The study was approved by the ethics committees at the University Hospital Motol and University Hradec Kralove in accordance with Czech law.
Consent for publication
LG provides remunerated consulting to OmniSEQ (Buffalo, NY, USA), Astra Zeneca (Gaithersburg, MD, USA), VL47 (New York, NY, USA) and the Luke Heller TECPR2 Foundation (Boston, MA, USA). All other authors have no financial interests to disclose.
- 34.Muth C, Rubner Y, Semrau S, Ruhle PF, Frey B, Strnad A, et al. Primary glioblastoma multiforme tumors and recurrence : Comparative analysis of the danger signals HMGB1, HSP70, and calreticulin. Strahlentherapie und Onkologie : Organ der Deutschen Rontgengesellschaft [et al]. 2016;192:146–55.CrossRefGoogle Scholar
- 37.Truxova I, Kasikova L, Salek C, Hensler M, Lysak D, Holicek P, Bilkova P, Holubova M, Chen X, Mikyskova R, Reinis M, Kovar M, Tomalova B, Kline J, Galluzzi L, Spisek R, Fucikova J. Calreticulin exposure on malignant blasts correlates with improved NK cell-mediated cytotoxicity in AML patients. Haematologica. 2019; in press.Google Scholar
- 38.Fucikova J, Kline J, Galluzzi L, Spisek R. Calreticulin arms NK cells against leukemia. Oncoimmunology. 2019; In press.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.