癌症生物学

A rigorous determination of effector contributions of tumor-infiltrating immune cells is critical for identifying targetable molecular mechanisms for the development of novel cancer immunotherapies. A tumor/immune cell–admixture model is an advantageous strategy to study tumor immunology as the fundamental methodology is relatively straightforward, while also being adaptable to scale to address increasingly complex research queries. Ultimately, this method can provide robust experimental information to complement more traditional murine models of tumor immunology. Here, we describe a tumor/macrophage-admixture model using bone marrow–derived macrophages to investigate macrophage-dependent tumorigenesis. Additionally, we provide commentary on potential branch points for optimization with other immune cells, experimental techniques, and cancer types.

Genome-editing technologies, especially CRISPR (clustered regularly interspaced short palindrome repeats)/Cas9 (CRISPR-associated protein 9), endows researchers the ability to make efficient, simple, and precise genomic DNA changes in many eukaryotic cell types. CRISPR/Cas9-mediated efficient gene knockout holds huge potential to improve the efficacy and safety of chimeric antigen receptor (CAR) T cell-based immunotherapies. Here, we describe an optimized approach for a complete loss of endogenous T cell receptor (TCR) protein expression, by CRISPR/Cas9-mediated TCR α constant (TRAC) and TCR β constant (TRBC) gene knockout, followed by subsequent CD3 negative selection in engineered human orthoCAR19 T cells. We believe this method can be expanded beyond CAR T cell application, and target other cell surface receptors.

Graphical abstract:

Schematic overview of the two-step process of endogenous TCR depletion in engineered human orthoCAR19 T cells using (1) CRISPR/Cas9-mediated gene knockout followed by (2) CD3 negative selection.

Transforming growth factor beta (TGF-β) is a multi-functional cytokine that plays a significant role in multiple diseases, including fibrosis and tumor progression. Whilst the biologic effects of TGF-β are well characterized, it is unclear how TGF-β signaling is regulated to impart specific responses within certain cell types. One mechanism of regulation may be through TGF-β activation, since TGF-β is always expressed in a latent form (L-TGF-β). Campbell et al. recently presented a new structural model to demonstrate how the integrin α_vβ₈ might specifically control TGF-β activation and signaling. In this model, α_vβ₈ binds to cell surface L-TGF-β1 to induce a conformational change, which exposes mature TGF-β peptide to TGF-β receptors (TGF-βRs), allowing initiation of TGF-β signaling from within the latent complex. This model also predicts that TGF-β signaling would be directed specifically towards the TGF-β expressing cell surface. We sought to test the validity of the new structural model by creating a cell-based assay which utilizes luciferase TGF-β reporter cells (TMLC). TMLC cells express high levels of TGF-βRs, but do not express cell surface L-TGF-β. We modified TMLC reporter cells to express cell surface L-TGF-β1 in a mutant form, which prevents the release of mature TGF-β from the latent complex. The newly generated cell lines were then used in a novel functional assay to investigate whether integrin α_vβ₈ could potentiate cell intrinsic TGF-β signaling from within the latent complex in vitro.

Nanomaterials are increasingly used for the diagnosis and treatment of cancer, including lung cancer. For the clinical translation of nano-based theranostics, it is vital to detect and monitor their accumulation in the tumor, as well as their interaction with tumor, immune cells, and the tumor microenvironment (TME). While high resolution microscopy of fixed tumor specimens can provide some of this information from individual thin slices, it cannot capture cellular events over time and lacks 3D information of the tumor tissue. On the other hand, in vivo optical procedures either fall short of providing the necessary cellular resolution, as in the case of epifluorescence optical imaging, or are very demanding, as for instance intravital lung microscopy. We describe an alternative approach to investigate nanoparticle-cell interactions in entire mouse lung lobes, by longitudinal live cell confocal microscopy at nanometer resolution. By filling the lung ex vivo with 1% agarose, we were able to stabilize the lung lobes and visualize the interaction of fluorescent cells and nanoparticles for at least 4 hours post mortem. This high resolution ex vivo live cell imaging approach is an easy 4D tool for assessing several dynamic processes in tumor tissue, such as the traffic of cells, shedding of extracellular vesicles (EVs), and the accumulation of nanoparticles in tumor tissue.

Graphic abstract:

Schematic of the workflow for live cell imaging in the mouse lung.

Flow cytometry is a popular laser-based technology that allows the phenotypic and functional characterization of individual cells in a high-throughput manner. Here, we describe a detailed procedure for preparing a single-cell suspension from mammary tumors of the mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) and analyzing these cells by multi-color flow cytometry. This protocol can be used to study the following tumor-infiltrating immune cell populations, defined by the expression of cell surface molecules: total leukocytes, tumor-associated macrophages (TAMs), conventional dendritic cells (DCs), CD103-expressing DCs, tumor-associated neutrophils, inflammatory monocytes, natural killer (NK) cells, CD4⁺ T cells, CD8⁺ T cells, γδT cells, and regulatory T cells.

Activating the STING (stimulator of interferon genes) signaling pathway via administration of STING agonist cyclic GMP-AMP (cGAMP) has shown great promise in cancer immunotherapy. While state-of-the-art approaches have predominantly focused on the encapsulation of cGAMP into liposomes or polymersomes for cellular delivery, we discovered that the recombinant STING protein lacking the transmembrane domain (STINGΔTM) could be used as a functional carrier for cGAMP delivery and elicit type I IFN expression in STING-deficient cell lines. Using this approach, we generated anti-tumoral immunity in mouse melanoma and colon cancer models, providing a potential translatable platform for STING agonist-based immunotherapy. Here, we report the detailed in vitro STING activation protocols with cGAMP-STINGΔTM complex to assist researchers in further development of this approach. This protocol can also be easily expanded to other applications related to STING activation, such as control of various types of infections.

Three-dimensional (3D) tumor spheroids have the potential to bridge the gap between two-dimensional (2D) monolayer tumor cell cultures and solid tumors with which they share a significant degree of similarity. However, the progression of solid tumors is often influenced by the dynamic and reciprocal interactions between tumor and immune cells. Here we present a 3D tumor spheroid-based model that might shed new light on understanding the mechanisms of tumor and immune cell interactions. The model first utilizes the hanging drop assay, which serves as one of the simplest methods for generating 3D spheroids and requires no specialized equipment. Next, pre-established spheroids can be co-cultured either directly or indirectly with an immune cell population of interest. Using skin melanoma, we provide a detailed description of the model, which might hold a significant importance for the development of successful therapeutic strategies.

This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live co-culture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five parts: (A) In silico characterization of the gene-of-interest (GOI) and protein-of-interest (POI); (B) design of the expression vector; (C) assembly of the expression vector; (D) transfection of a eukaryotic cell line with the expression vector; (E) testing of the recombinant protein. This extensive protocol can be completed with only polymerase chain reaction (PCR) and cell culture training. Additionally, each part of the protocol can be used independently.

The binding interactions of PD-1 and PD-L1 have been studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) over the past few years, but these investigations resulted in controversy regarding the values of the dissociation constant (K_d) (Freeman et al., 2000). MST is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. The technique is based on the movement of molecules along microscopic temperature gradients, and it detects changes in their hydration shell, charge or size. One binding partner is fluorescently labeled, while the other binding partner remains label-free. We used a protocol that allows the determination of the binding affinity by MST without purification of the target protein from the cell lysate. The application of this MST method to PD-1-eGFP and PD-L1-eGFP expressed in CHO-K1 cells allowed us, for the first time, to determine the affinity of the complex formed between PD-1 and its ligand PD-L1 during tumor escape. The protocol has a variety of potential applications for studying the interactions of proteins with small molecules.

Intranasal administration of vaccine adjuvants directly deliver therapeutic agents to the lungs to induce potent lung mucosal immune responses. Cyclic di-GMP (CDG) is a promising mucosal vaccine adjuvant candidate capable of inducing protective immunity. This protocol describes an in vivo approach to induce and detect mucosal (lung) and systemic (blood and spleen) vaccine adjuvant responses of CDG. This protocol also includes the methods to detect both humoral and cellular immune responses of CDG adjuvant. Last, this protocol can be used to study other cyclic dinucleotides as mucosal vaccine adjuvants.