Plasmacytoid dendritic cells (pDCs) are a minority subset of dendritic cells that despite their tiny quantity play an important role in the immune system, especially in antiviral immunity. They are known mostly as the major producers of type I IFN, which they secrete upon stimulation of endosomal Toll-like receptors 7 and 9 with viral RNA and DNA. However, the functionality of pDCs is more complex, as they were shown to be also involved in autoimmunity, inflammation, and cancer. In the context of the tumor microenvironment, pDCs mostly show substantial functional defects and thus contribute to establishing immunosuppressive micromilieu. Indeed, tumor-infiltrating pDCs were shown to be predominantly pro-tumorigenic, with reduced ability to produce IFNα and capacity to prime regulatory T cells via the ICOS/ICOS-L pathway. Here we describe in detail a method to assess the functional capacity of pDCs upon exposure to tumor-derived cell culture supernatants. The same technique can be implemented with minimal variations to test any soluble factor's impact on pDC phenotype and function.

Alloreactive T-cell responses against mismatched MHC or minor histocompatibility antigens may result in deleterious graft-versus-host disease (GVHD) and increased morbidity and mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, these T-cell responses may be directed against residual tumor cells (the graft-versus-tumor effect, GVT), thus preventing relapse of the disease. Recent findings have shown that CD45RA naïve T cells, but not CD45RA memory T cells are the major contributors to GVHD, thus leading to clinical trials where CD45RA-depleted, memory-enriched T-cell products are adoptively transferred following allo-HSCT to prevent GVHD and enhance immune reconstitution. However, residual alloreactivity may still be present in the memory T-cell compartment, thus contributing to prevent disease relapse by GVT. Here, we describe a simple cell-based protocol to identify alloreactive naïve and memory T cells by co-culturing T-cell subsets and third-party antigen-presenting cells. The responding cells are identified following dilution of carboxyfluorescein succinimidyl ester (CFSE) and upregulation of the activation marker CD25. These CFSE-diluting cells can be further phenotyped by high-dimensional flow cytometry, or purified with a cell sorter for downstream genomic and functional assays.

Myeloid-derived suppressor cells (MDSCs) encompass a diverse population of immature myeloid cells categorized into granulocytic and monocytic groups. These cells exert immune-suppressive functions within the tumor microenvironment, primarily influenced by cytokines and tumor-associated factors. Research has consistently linked elevated MDSC levels to unfavorable cancer prognosis and poor responses to immunotherapies. Here, we detail the materials, equipment, and methods involved in MDSC analysis in human peripheral blood by flow cytometry, emphasizing the importance of selecting appropriate antibody clones and fluorochromes for precise cell population discrimination. The gating strategy is described, with particular attention to the challenges associated with defining conjugated antibody labeling positive and negative populations.

Adoptive natural killer (NK) cell-based immunotherapy is a promising treatment approach in cancer that is showing notable efficacy against hematological malignancies. However, the success of NK cell immunotherapy in patients with solid tumors is limited due to several barriers, which include the immunosuppressive tumor microenvironment (TME), heterogeneity of tumor cells and poor NK cell infiltration into the tumor. Advances in 3D in vitro culture technologies have opened new avenues for the development of more physiological human cancer models that mimic important tumor features which are absent in traditional 2D studies and may be essential for the improvement of immunotherapies against solid tumors. Here, we describe a comprehensive protocol to generate tumor spheroids from the A549 lung carcinoma cell line, then establish co-cultures with NK cells to, ultimately, determine NK cell functional response with a degranulation assay, a surrogate of NK cell cytotoxicity against tumor spheroids. Additionally, we studied degranulation by stimulating NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) with cetuximab, an IgG1 monoclonal antibody used in cancer therapy. Likewise, other monoclonal antibodies or combination treatments could also be studied in this 3D co-culture system, providing very valuable information to define effective combinations of therapeutic agents able to generate NK cells with high cytotoxic potential that could lead to more successful adoptive NK cell-based therapies for the treatment of solid tumors.

Multiparametric flow cytometry (MFC) represents an essential tool for immune monitoring, and validation of MFC panels is a fundamental prerequisite in routine laboratory settings as well as for translational and clinical research purposes. Regulatory T cells (TREGs) constitute a subset of CD4+ effector T cells that modulate the immune response in numerous settings, including autoimmune disease, allergy, microbial infection, tumor immunity, transplantation, and more. These cells comprise a small fraction of total CD4+ T cells in human peripheral blood and mouse spleen. In oncology, TREG cells are highly relevant, as they are involved in the suppression of the anti-tumor response in many types of cancer, to the extent that the first immune checkpoint inhibitor approved for clinical use in humans was a monoclonal antibody directed against CTLA-4, a molecule functionally associated with TREGs. Due to all these factors, robust assays are mandatory to accurately determine TREG cell frequency and function. Here, we describe the validation of an 8-color flow-cytometry protocol for TREG detection and analysis in a real-world laboratory scenario. The entire process includes the workflow plan and the standard operating procedure resembling each phase, from the panel design to the staining, acquisition, and analysis steps. Validation is planned to be performed in replicates on fresh whole blood samples derived from multiple healthy subjects. The analytical validity of the TREG cell assay is ensured by testing the intra-assay accuracy. The detailed procedure for the entire process is accompanied by important troubleshooting suggestions and other useful tips.

Antibodies directed against surface antigens of tumor cells are commonly found in sera of cancer patients and of oncological animal models. Polyclonal antibodies directed against various epitopes of the same antigen may be spontaneously elicited by tumor antigens or may result from the administration of specific vaccines and other immunostimulating treatments. Furthermore, after therapeutic administration of monoclonal antibodies, the antibody will be detectable in the bloodstream for several weeks. Circulating antibodies are easily detected with enzyme-linked immunosorbent assays (ELISA) and other immunometric tests which, however, cannot tell whether the antibodies are functional, i.e. whether they can significantly inhibit (or enhance) tumor growth. One possibility would be to treat conventional (i.e. bi-dimensional, 2D) tumor cell cultures with antibody-containing sera. However, in several instances, it was found that 2D cultures were poorly sensitive, even to powerful monoclonal antibodies like trastuzumab, whereas three-dimensional (3D) cultures may better reveal the tumor-inhibitory activity of circulating antibodies. We describe here a breast cancer 3D soft agar colony growth inhibition assay that was developed to quantify the tumor cell inhibitory activity of antibodies against human HER-2 elicited in mice by specific vaccines. The assay might be readily modified to analyze antibodies against different surface antigens expressed by other tumor types and also for testing of new monoclonal antibodies and nanobodies.

At odds with historical views suggesting that mitochondrial functions are largely dispensable for cancer cells, it is now clear that mitochondria have a major impact on malignant transformation, tumor progression and response to treatment. Mitochondria are indeed critical for neoplastic cells not only as an abundant source of ATP and other metabolic intermediates, but also as gatekeepers of apoptotic cell death and inflammation. Interestingly, while mitochondrial components are mostly encoded by nuclear genes, mitochondria contain a small, circular genome that codes for a few mitochondrial proteins, ribosomal RNAs and transfer RNAs. Here, we describe a straightforward method to generate transmitochondrial cybrids, i.e., cancer cells depleted of their mitochondrial DNA and reconstituted with intact mitochondria from another cellular source. Once established, transmitochondrial cybrids can be stably propagated and are valuable to dissect the specific impact of the mitochondrial genome on cancer cell functions.

Cellular senescence is a damage-induced condition characterized by enduring cell cycle arrest and a heightened secretory profile known as the senescence-associated secretory phenotype (SASP). The SASP consists not only of release of inflammatory cytokines and chemokines that attract and activate a diverse repertoire of innate and adaptive immune cells, but also the upregulation of immunomodulatory cell surface molecules that promote immune clearance of senescent cells. Natural Killer (NK) cells are particularly adept at sensing and eliminating senescent cells. In the setting of cancer, commonly administered cytotoxic and cytostatic therapies can elicit senescence and in turn reactivate NK cell immune surveillance against tumors. Here, we detail a series of in vivo, ex vivo, and in vitro assays to assess the impact of therapy-induced senescence on NK cell phenotypes, including their activation, exhaustion, migration, and killing capacity in the context of pancreatic cancer. Importantly, this methodology can be adapted to investigate NK cell biology across various disease states and treatment modalities and help inform NK cell-based immunotherapies for cancer.

Myeloid-derived suppressor cells (MDSCs) are cells that play a regulatory role in immune responses and inflammation. They can have both positive and negative effects on various diseases, including cancer, infections, sepsis, and trauma. MDSCs inhibit immune cells by releasing immunosuppressive factors and can be categorized as monocytic (M) or polymorphonuclear (PMN) cell lineages. Most MDSCs are PMN-MDSC and are found in the peripheral blood (PB) and in the tissue microenvironment of tumor and inflamed patients, where they can directly inhibit immune cell activity and promote tumor progression. Various markers have been suggested for their identification, but in order to be defined as MDSC, their inhibitory capacity has to be certified. In this article, we summarize the identification and functional protocol for characterizing MDSCs, focusing on PMN-MDSC.

Natural Killer (NK) cells are cytotoxic lymphocytes involved in the recognition of pathogen-infected and cancer cells. NK cells are very attractive as cell therapy tools because they are neither restricted by donor compatibility nor do they cause toxicity. Although their anti-tumor role has been long known, for development of NK-based therapies it is important to select the appropriate subpopulation. Similarly, non-MHC restricted T cells, in particular γδ T cells, have also been proposed as novel weapons against cancer. Here, we describe a new approach for production and characterization of anti-tumor innate lymphocyte cultures, containing mainly NK and γδ T cells, based on stimulation of peripheral blood mononuclear cells (PBMC) with BCG (Bacillus Calmette-Guérin), the tuberculosis vaccine, which is also successfully used to treat non-muscle invasive bladder cancer. Anti-tumor innate lymphocytes specifically proliferate from BCG-primed PBMC and can be cultured for weeks in low doses of IL12, IL15 and IL21. These cells kill a wide range of tumors and remain functional for weeks, with minimal manipulation. The phenotypic analysis of these cultures by multi-parametric flow cytometry is explained. Functional assays, including lymphocyte degranulation, cytokine production and radioactive isotope-free specific lysis experiments are also described.

Brain metastases (BrM) occur when malignant cells spread from a primary tumor located in other parts of the body to the brain. BrM is a deadly complication for cancer patients and severely lacks effective therapies. Due to the limited access to patient samples, preclinical models remain a very valuable tool for studying metastasis development, progression, and response to therapy. Thus, reliable methods to assess metastatic burden in these models are crucial. Here we describe step by step a new semi-automatic machine-learning approach to quantify metastatic burden on mouse whole-brain stereomicroscope images while preserving tissue integrity. This protocol uses the open-source and user-friendly image analysis software QuPath. The method is fast, reproducible, unbiased, and gives access to data points not always accessible with other existing strategies.

Immunogenic cell death (ICD) has emerged as a pivotal form of cell death in anti-cancer therapy as it combines the ability to both eliminate cancer cells and simultaneously activate anti-tumor immunity, thereby contributing to the establishment of long-term immunological memory. Antigen-presenting cells (APCs), with an emphasis on dendritic cells (DCs), play a central role in bridging the innate and adaptive immune systems. DCs recognize and present antigens derived from the dying cancer cells to T cells in the lymph nodes, resulting in T cell activation. The activation and maturation of DCs thus marks the initiation of a cycle of anti-tumor immunity. In this chapter, we provide straightforward methodologies to isolate DCs from murine bone marrow (bone marrow-derived DCs, BMDCs), induce immunogenic apoptosis in murine MCA205 fibrosarcoma cells using ICD inducer mitoxantrone (MTX), co-cultivate BMDCs with the MTX-treated cancer cells, and to assess the activation and maturation status of BMDCs by flow cytometric-assisted quantification of co-stimulatory molecules (MHC II, CD86, CD80) expressed on the plasma membrane of BMDCs. With minor adjustments, the same protocol can be implemented to other cancer cell lines or to analyze the phenotypic status of non-professional APCs.

Personalized immunotherapy is emerging as a promising approach for cancer treatment, aiming to harness the patient's own immune system to target and eliminate tumor cells. One key aspect of developing effective personalized immunotherapies is the utilization of tumor slices derived from individual patient tumors. Tumor slice models retain the complexity and heterogeneity of the original tumor microenvironment, including interactions with immune cells, stromal elements, and vasculature. These ex vivo models serve as valuable tools for studying tumor-immune interactions and for testing the efficacy of immunotherapeutic agents tailored to the specific characteristics of each patient's tumor. In this chapter, we set up a protocol for immunotherapy strategies in mouse models highlighting their translational potential to guide treatment decisions and improve therapeutic outcomes in cancer patients.

Colorectal cancer (CRC) presents a substantial global health challenge, prompting the necessity for the development and validation of preclinical models to enhance our comprehension and therapeutic interventions. Among the myriad of murine models available for CRC evaluation, orthotopic implantation via intercaecal microinjection stands out as a preferred method for replicating the intricate tumor microenvironment while ensuring uniformity and standardized applicability. In this study, we delineate a methodology addressing the required steps for tumor cell line selection and reporter transduction, animal model preparation, orthotopic tumor implantation, in vivo monitoring of tumor growth and metastasis formation. We comprehensively describe the generation of a xenograft murine model based on the intercaecal implantation of human GFP/luciferase SW620 CRC cells, facilitating the evaluation of responses to pre-clinical human-based therapeutic approaches. The implementation of these standardized protocols promises to augment the reliability and reproducibility of preclinical studies, ultimately advancing our comprehension of CRC pathogenesis and guiding the development of innovative therapeutic strategies.

To develop new effective therapeutic strategies for cancer patients, there is a need for extensive and precise insights into the mechanisms involved in the immune response to anti-cancer treatments. The enzyme-linked immunospot (ELISpot) assay is a rapid and reproducible technique that allows the detection of cytokine-producing antigen-specific T cells at the single cell level. This protocol describes an interferon gamma (IFN-γ) ELISpot method for measuring antigen-specific murine CD8 T cells that produce IFN-γ, a marker of their activation and cytotoxicity. Splenocytes from tumor-bearing mice treated with radiation therapy were used as source of CD8 T cells and were stimulated with a tumor-derived peptide. This method was facilitated by a ready-to-use assay kit and provides a tool to analyze the specificity, intensity, and kinetics of specific CD8 T cells.

Immune checkpoint blockade-based cancer immunotherapy is an effective tool for cancer treatment. PD-1/PD-L1 blockade, however, is limited by a low response rate and adaptive resistance. A growing body of studies has shown that the high stromal content dense with extracellular matrix plays a significant role in immune checkpoint blockade resistance as well as T cell exclusion. In addition to physically obstructing immune cell infiltration, the extracellular matrix (ECM) may also interact with T cell receptors to indirectly impair their effector function and lead to anti-PD-1 resistance. Anti-PD-1 resistance may thus be overcome by rupturing the physical barrier related negative immune regulation, which may improve T cell infiltration and the efficacy of cancer immunotherapy. Here, we offer two straightforward methods based on flow cytometry and confocal microscopy to evaluate the effectiveness of an inhibitor targeting the novel "stromal checkpoint" DDR1/collagen, which aims to facilitate T cell migration and infiltration of tumor spheres by overcoming collagen barriers. With minor variations, the same method can be easily modified to test the inhibitors that target other immune checkpoints, and the extracellular matrix-associated drug targets that mediate anti-PD-1 resistance.

Exposure of the endoplasmic reticulum chaperone calreticulin (CALR) on the surface of stressed and dying cells is paramount for their effective engulfment by professional antigen-presenting cells such as dendritic cells (DCs). Importantly, this is required (but not sufficient) for DCs to initiate an adaptive immune response that culminates with an effector phase as well as with the establishment of immunological memory. Conversely, the early exposure of phosphatidylserine (PS) on the outer layer of the plasma membrane is generally associated with the rapid engulfment of stressed and dying cells by tolerogenic macrophages. Supporting the clinical relevance of the CALR exposure pathway, the spontaneous or therapy-driven translocation of CALR to the surface of malignant cells, as well as intracellular biomarkers thereof, have been associated with improved disease outcome in patients affected by a variety of neoplasms, with the notable exception of multiple myeloma (MM). Here, we describe an optimized protocol for the flow cytometry-assisted quantification of surface-exposed CALR and PS on CD38 plasma cells from the bone marrow of patients with MM. With some variations, we expect this method to be straightforwardly adaptable to the detection of CALR and PS on the surface of cancer cells isolated from patients with neoplasms other than MM.

Colorectal cancer (CRC) research demands reliable experimental models to enhance translational potential. Immortalized cancer cell lines, although commonly employed, exhibit limitations such as phenotypic divergence from primary tumors, which underscores the need for more representative models. This method chapter presents a protocol for collecting and processing primary CRC specimens for in vitro assays to assess the cytotoxic potential of antitumor agents, with a focus on adoptive cellular therapies. The protocol emphasizes the importance of immediate processing to minimize ex vivo alterations and includes guidelines for cryopreservation, thawing, enzymatic digestion, and mechanical disruption, which were optimized for increased cell yield and viability. An optional step of immune cell depletion is included to avoid indirect effects of endogenous leukocytes, with the option to retain this fraction for further analysis. Finally, the steps for flow cytometry-based evaluation of tumor cell apoptosis by assessment of Caspase 3/7 staining are detailed. The implementation of this standardized protocol using patient-derived specimens offers a superior alternative to immortalized cell lines for assessing therapeutic efficacy, increasing the probability of translation of preclinical research findings, and bolstering the development of innovative therapeutic strategies for CRC.

Dendritic cells (DCs), and especially so conventional type I DCs (cDC1s), are fundamental regulators of anticancer immunity, largely reflecting their superior ability to engulf tumor-derived material and process it for cross-presentation on MHC Class I molecules to CD8 cytotoxic T lymphocytes (CTLs). Thus, investigating key DC functions including (but not limited to) phagocytic capacity, expression of CTL-activating ligands on the cell surface, and cross-presentation efficacy is an important component of multiple immuno-oncology studies. Unfortunately, DCs are terminally differentiated cells, implying that they cannot be propagated indefinitely in vitro and hence must be generated ad hoc from circulating or bone marrow-derived precursors, which presents several limitations. Here, we propose a simple, cytofluorometric method to quantify phenotypic activation markers including CD80, CD86 and MHC class II molecules on the surface of a conditionally immortalized immature DC line that can be indefinitely propagated in vitro but also driven into maturation at will with a simple change in culture conditions. Upon appropriate scaling and automatization, this approach is compatible with high-throughput screening programs for the discovery of novel DC activators that do not suffer from batch variability and other limitations associated with the generation of fresh DCs.

The immune compartment of a tissue is dynamic, changing to respond to infections, tumors, or therapeutic interventions. Within tissues, local microenvironments provide interaction partners and cytokines that can gear immune cells into distinct functional states. Thus, it is not just the immune composition of a tissue, but also the relative localization of immune cells that determines the outcome of a response. Conventional techniques like immunohistochemistry (IHC) have been used to describe infiltration of immune cells and their relative position within tissues. However, these technologies are limited on the number of targets that can be simultaneously imaged. Here, we describe a simple protocol using imaging mass cytometry (IMC) for immunophenotyping formalin-fixed, paraffin-embedded (FFPE) tissues. IMC has a 1-μm resolution and allows simultaneous detection of up to 40 targets, overcoming limitations of traditional methods. In this protocol, we detail the staining procedure, offer an example of a murine FFPE antibody panel for immunophenotyping, and additionally provide suggestions for initial image analysis. The herein presented workflow facilitates the characterization of immune niches and can be used to assess their alterations throughout immune responses or therapeutic interventions. With minimal alterations, this approach can be used on clinically relevant samples or animal models to investigate specific immune responses and better understand disease progression or treatment dynamics.