Cancer Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 1107 Views Nov 20, 2023

Cancer cells evade the immune system by downregulating antigen presentation. Although immune checkpoint inhibitors (ICI) and adoptive T-cell therapies revolutionized cancer treatment, their efficacy relies on the intrinsic immunogenicity of tumor cells and antigen presentation by dendritic cells. Here, we describe a protocol to directly reprogram murine and human cancer cells into tumor-antigen-presenting cells (tumor-APCs), using the type 1 conventional dendritic cell (cDC1) transcription factors PU.1, IRF8, and BATF3 delivered by a lentiviral vector. Tumor-APCs acquire a cDC1 cell-like phenotype, transcriptional and epigenetic programs, and function within nine days (Zimmermannova et al., 2023). Tumor-APCs express the hematopoietic marker CD45 and acquire the antigen presentation complexes MHC class I and II as well as co-stimulatory molecules required for antigen presentation to T cells, but do not express high levels of negative immune checkpoint regulators. Enriched tumor-APCs present antigens to Naïve CD8+ and CD4+ T cells, are targeted by activated cytotoxic T lymphocytes, and elicit anti-tumor responses in vivo. The tumor-APC reprogramming protocol described here provides a simple and robust method to revert tumor evasion mechanisms by increasing antigen presentation in cancer cells. This platform has the potential to prime antigen-specific T-cell expansion, which can be leveraged for developing new cancer vaccines, neoantigen discovery, and expansion of tumor-infiltrating lymphocytes.

Key features

• This protocol describes the generation of antigen-presenting cells from cancer cells by direct reprogramming using lineage-instructive transcription factors of conventional dendritic cells type I.

• Verification of reprogramming efficiency by flow cytometry and functional assessment of tumor-APCs by antigen presentation assays.

0 Q&A 678 Views Jul 5, 2023

The rapid display and delivery method for customized tumor mRNA vaccines is limited. Herein, bacteria-derived outer membrane vesicles (OMVs) are employed as an mRNA delivery platform by surface engineering of an RNA-binding protein, L7Ae. OMV-L7Ae can rapidly adsorb boxC/D sequence-labeled mRNA antigens through L7Ae-boxC/D binding and deliver them into HEK-293T and dendritic cells. This platform provides an mRNA delivery technology distinct from lipid nanoparticles (LNPs) for personalized mRNA tumor vaccination and with a Plug-and-Display strategy suitable for rapid preparation of the personalized mRNA tumor vaccine against varied tumor antigens.

Key features

• OMVs are employed as an mRNA delivery platform through L7Ae-boxC/D binding.

Graphical overview

0 Q&A 1280 Views Jun 5, 2023

Exosomes are lipid bilayer–enclosed vesicles, actively secreted by cells, containing proteins, lipids, nucleic acids, and other substances with multiple biological functions after entering target cells. Exosomes derived from NK cells have been shown to have certain anti-tumor effects and potential applications as chemotherapy drug carriers. These developments have resulted in high demand for exosomes. Although there has been large-scale industrial preparation of exosomes, they are only for generally engineered cells such as HEK 293T. The large-scale preparation of specific cellular exosomes is still a major problem in laboratory studies. Therefore, in this study, we used tangential flow filtration (TFF) to concentrate the culture supernatants isolated from NK cells and isolated NK cell–derived exosomes (NK-Exo) by ultracentrifugation. Through a series of characterization and functional verification of NK-Exo, the characterization, phenotype, and anti-tumor activity of NK-Exo were verified. Our study provides a considerably time- and labor-saving protocol for the isolation of NK-Exo.

0 Q&A 511 Views May 20, 2023

P18F3-based bi-modular fusion proteins (BMFPs), designed to re-direct pre-existing anti-Epstein-Barr virus (EBV) endogenous polyclonal antibodies towards defined target cells, demonstrated efficient biological activity in a mouse tumor model and could potentially represent a universal and versatile platform to develop novel therapeutics against a broad range of diseases. This protocol provides step-by-step instructions for expressing scFv2H7-P18F3, a BMFP targeting human CD20, in Escherichia coli (SHuffle®), and for purifying soluble proteins using a two-step process, namely immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography. This protocol can also be used for expression and purification of other BMFPs with alternative binding specificities.

0 Q&A 856 Views Mar 5, 2023

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.

0 Q&A 1955 Views Aug 5, 2022

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.

0 Q&A 2052 Views Apr 20, 2022

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 αvβ8 might specifically control TGF-β activation and signaling. In this model, αvβ8 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 αvβ8 could potentiate cell intrinsic TGF-β signaling from within the latent complex in vitro.

0 Q&A 1861 Views Jan 20, 2022

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.

1 Q&A 5100 Views Jun 5, 2021

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.

0 Q&A 4370 Views Feb 5, 2021
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.

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