Immunogenic cell death (ICD), a functionally peculiar type of apoptosis, represents a unique way to deliver danger-associated molecular patterns (DAMPs) to the tumor microenvironment. Once emitted by dying cancer cells, DAMPs orchestrate antigen-specific immune responses by acting on both innate and adaptive components of the immune system. Accumulating preclinical and clinical evidence indicates that one of these DAMPs, calreticulin (CALR) represents a novel powerful prognostic biomarker, reflecting the activation of a clinically relevant anticancer immune response in different cancer malignancies. Therefore, the assessment of CALR emission can provide a therapeutic tool for the stratification of cancer patients and the identification of individuals that are intrinsically capable to respond to a particular treatment. Here we describe methods for the quantification of CALR exposure in the tumor microenvironment of cancer patients by flow cytometry and immunohistochemistry.

• MeSH
• Immunogenic Cell Death * MeSH
• Immunohistochemistry methods   MeSH
• Calreticulin analysis   immunology   MeSH
• Humans MeSH
• Biomarkers, Tumor analysis   immunology   MeSH
• Tumor Microenvironment MeSH
• Neoplasms immunology   pathology   MeSH
• Flow Cytometry methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

Natural killer (NK) cells constitute the predominant innate lymphocyte subset that mediates the anti-viral and anti-tumor immune responses. NK cells use an array of innate receptors to sense their environment and to respond to infections, cellular stress and transformation. The resulting NK cell activation, including cytotoxicity and cytokine production, is a fundamental component of the early immune response. The most recent discoveries in NK cell biology have stimulated the translational research that has led to remarkable results for the treatment of human malignancies. Therefore, the rapid isolation of NK cells from the peripheral blood or tumor microenvironment and the subsequent assessment of cytolytic function are crucial to the study of their potency and NK cell-mediated immunosurveillance. Here, we provide protocols for NK cell isolation and the assessment of NK cell cytotoxicity using flow cytometry.

• MeSH
• Lymphocyte Activation MeSH
• Killer Cells, Natural immunology   MeSH
• Cytotoxicity, Immunologic * MeSH
• Cytotoxicity Tests, Immunologic methods   MeSH
• Humans MeSH
• Flow Cytometry methods   MeSH
• Cell Separation methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: 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. METHOD: 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. RESULTS: 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. CONCLUSIONS: 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.

• MeSH
• Adult MeSH
• Calreticulin immunology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Tumor Microenvironment genetics   immunology   MeSH
• Ovarian Neoplasms genetics   immunology   MeSH
• Prognosis MeSH
• RNA-Seq MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Endoplasmic Reticulum Stress MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Cancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called "damage-associated molecular patterns," DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed CRT correlated with an increased proportion of natural killer cells and effector memory CD4(+) and CD8(+) T cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response.

• MeSH
• Leukemia, Myeloid, Acute drug therapy   genetics   immunology   metabolism   MeSH
• Alarmins metabolism   MeSH
• Blast Crisis immunology   pathology   MeSH
• Cell Death MeSH
• CD8-Positive T-Lymphocytes immunology   MeSH
• Phenotype MeSH
• Transcription, Genetic MeSH
• Immunity MeSH
• Calreticulin metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Multivariate Analysis MeSH
• Proportional Hazards Models MeSH
• HSP70 Heat-Shock Proteins metabolism   MeSH
• HSP90 Heat-Shock Proteins metabolism   MeSH
• Gene Expression Regulation, Leukemic MeSH
• Gene Expression Profiling MeSH
• Th1 Cells immunology   MeSH
• Treatment Outcome MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Progressing malignancies establish robust immunosuppressive networks that operate both systemically and locally. In particular, as tumors escape immunosurveillance, they recruit increasing amounts of myeloid and lymphoid cells that exert pronounced immunosuppressive effects. These cells not only prevent the natural recognition of growing neoplasms by the immune system, but also inhibit anticancer immune responses elicited by chemo-, radio- and immuno therapeutic interventions. Throughout the past decade, multiple strategies have been devised to counteract the accumulation or activation of tumor-infiltrating immunosuppressive cells for therapeutic purposes. Here, we review recent preclinical and clinical advances on the use of small molecules that target the immunological tumor microenvironment for cancer therapy. These agents include inhibitors of indoleamine 2,3-dioxigenase 1 (IDO1), prostaglandin E2, and specific cytokine receptors, as well as modulators of intratumoral purinergic signaling and arginine metabolism.

• Publication type
• Journal Article MeSH
• Review MeSH

A high density of tumor-infiltrating mature dendritic cells (DC) and CD8(+) T cells correlates with a positive prognosis in a majority of human cancers. The recruitment of activated lymphocytes to the tumor microenvironment, primed to recognize tumor-associated antigens, can occur in response to immunogenic cell death (ICD) of tumor cells. ICD is characterized by the preapoptotic translocation of calreticulin (CRT) from the endoplasmic reticulum (ER) to the cell surface as a result of an ER stress response accompanied by the phosphorylation of eukaryotic initiation factor 2α (eIF2α). We conducted a retrospective study on two independent cohorts of patients with non-small cell lung cancer (NSCLC) to investigate the prognostic potential of CRT. We report that the level of CRT expression on tumor cells, which correlated with eIF2α phosphorylation, positively influenced the clinical outcome of NSCLC. High CRT expression on tumor cells was associated with a higher density of infiltrating mature DC and effector memory T-cell subsets, suggesting that CRT triggers the activation of adaptive immune responses in the tumor microenvironment. Accordingly, patients with elevated CRT expression and dense intratumoral infiltration by DC or CD8(+) T lymphocytes had the best prognosis. We conclude that CRT expression constitutes a new powerful prognostic biomarker that reflects enhanced local antitumor immune responses in the lung.

• MeSH
• Calreticulin metabolism   MeSH
• Cohort Studies MeSH
• Middle Aged MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Lung Neoplasms metabolism   mortality   MeSH
• Carcinoma, Non-Small-Cell Lung metabolism   mortality   MeSH
• Prognosis MeSH
• Prospective Studies MeSH
• Retrospective Studies MeSH
• T-Lymphocyte Subsets metabolism   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

It is now clear that human neoplasms form, progress, and respond to therapy in the context of an intimate crosstalk with the host immune system. In particular, accumulating evidence demonstrates that the efficacy of most, if not all, chemo- and radiotherapeutic agents commonly employed in the clinic critically depends on the (re)activation of tumor-targeting immune responses. One of the mechanisms whereby conventional chemotherapeutics, targeted anticancer agents, and radiotherapy can provoke a therapeutically relevant, adaptive immune response against malignant cells is commonly known as "immunogenic cell death." Importantly, dying cancer cells are perceived as immunogenic only when they emit a set of immunostimulatory signals upon the activation of intracellular stress response pathways. The emission of these signals, which are generally referred to as "damage-associated molecular patterns" (DAMPs), may therefore predict whether patients will respond to chemotherapy or not, at least in some settings. Here, we review clinical data indicating that DAMPs and DAMP-associated stress responses might have prognostic or predictive value for cancer patients.

• Publication type
• Journal Article MeSH
• Review MeSH