Methods in cell biology最新文献

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.

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.

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.

Super-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targeted in situ structural studies of biological samples. By combining the high specificity and localization accuracy of single-molecule localization microscopy (cryoSMLM) with the high resolution of cryo-electron tomography (cryoET), this method enables accurately targeted data acquisition and the observation and identification of biomolecules within their natural cellular context. Despite its potential, the adaptation of SRcryoCLEM has been hindered by the need for specialized equipment and expertise. In this chapter, we outline a workflow for cryoSMLM and cryoET-based SRcryoCLEM, and we demonstrate that, given the right tools, it is possible to incorporate cryoSMLM into an established cryoET workflow. Using Vimentin as an exemplary target of interest, we demonstrate all stages of an SRcryoCLEM experiment: performing cryoSMLM, targeting cryoET acquisition based on single-molecule localization maps, and correlation of cryoSMLM and cryoET datasets using scNodes, a software package dedicated to SRcryoCLEM. By showing how SRcryoCLEM enables the imaging of specific intracellular components in situ, we hope to facilitate adoption of the technique within the field of cryoEM.

Molecular cytometry refers to a group of high-parameter technologies for single-cell analysis that share the following traits: (1) combined (multimodal) measurement of protein and transcripts, (2) medium throughput (10-100K cells), and (3) the use of oligonucleotide-tagged antibodies to detect protein expression. The platform can measure over 100 proteins and either hundreds of targeted genes or the whole transcriptome, on a cell-by-cell basis. It is currently one of the most powerful technologies available for immune monitoring. Here, we describe the technology platform (which includes CITE-Seq, REAP-Seq, and AbSeq), provide guidance for its optimization, and discuss advantages and limitations. Finally, we provide some vignettes from studies that demonstrate the application and potential insight that can be gained from molecular cytometry studies.

Multiple technologies have been used to monitor response to therapy in acute myeloid leukemia (AML) to improve detection of leukemia over the standard of practice, morphologic counting of blasts. The two techniques most frequently used in a routine clinical setting, flow cytometry and RQ-PCR, differ in their targets, sensitivity, and ability to detect residual disease. Both flow cytometry and RQ-PCR detect the expression of abnormal gene products, at the protein level or RNA level, respectively. Flow cytometry can be applied to a broad range of AML cases while RQ-PCR is limited to specific genetic abnormalities identified in subsets of AML. This article compares the results when both techniques were used in a reference laboratory to monitor AML over the course of treatment, comparing quantitative and qualitative results.

Correlative cryo-microscopy pipelines combining light and electron microscopy and tomography in cryogenic conditions (cryoCLEM) on the same sample are powerful methods for investigating the structure of specific cellular targets identified by a fluorescent tag within their unperturbed cellular environment. CryoCLEM approaches circumvent one of the inherent limitations of cryo EM, and specifically cryo electron tomography (cryoET), of identifying the imaged structures in the crowded 3D environment of cells. Whereas several cryoCLEM approaches are based on thinning the sample by cryo FIB milling, here we present detailed protocols of two alternative cryoCLEM approaches for in situ studies of adherent cells at the single-cell level without the need for such cryo-thinning. The first approach is a complete cryogenic pipeline in which both fluorescence and electronic imaging are performed on frozen-hydrated samples, the second is a hybrid cryoCLEM approach in which fluorescence imaging is performed at room temperature, followed by rapid freezing and subsequent cryoEM imaging. We provide a detailed description of the two methods we have employed for imaging fluorescently labeled cellular structures with thickness below 350-500nm, such as cell protrusions and organelles located in the peripheral areas of the cells.

Telomerase is a reverse transcriptase that consists of the telomerase reverse transcriptase (TERT) protein and the telomerase RNA component TERC which also harbors the template region for telomere synthesis. In its canonical function the enzyme adds single-stranded telomeric hexanucleotides de novo to the ends of linear chromosomes, telomeres, in telomerase-positive cells such as germline, stem- and cancer cells. This potential biochemical activity of telomerase can be measured with the help of a telomerase repeat amplification protocol (TRAP) which often includes a PCR amplification due to the low abundance of telomerase in most cells and tissues. The current chapter describes various TRAP methods to detect telomerase activity (TA) using gel-based methods, its advantages and deficits, how to perform an ELISA-based TRAP assay and how best to interpret its results. Since development of the TRAP assay in 1994, there have been numerous modifications and adaptations of the method from real-time PCR analysis, isothermal amplification and nanotechnology to CRISPR/Cas-based methods which will be briefly mentioned. However, it is not possible to cover all different TRAP methods and thus there is no comprehensiveness claimed by this chapter. Instead, the author describes various aspects of using TRAP assays including required controls, sample preparation, etc. in order to avoid pitfalls and set-backs in applying this rather complex and demanding technique. The TRAP assay is particularly important to support clinical diagnosis of cancer, analyze tumor therapy as well as to evaluate various approaches to inhibit TA as a form of anti-cancer therapy.

Tumor-draining lymph nodes (tumor-DLNs) provide a rich source of tumor-reactive lymphocytes which can be used in adoptive immunotherapy (AIT) and that circumvent the need to resect autologous tumor, without the challenges and shortcomings associated with using autologous tumor or anti-CD3 monoclonal antibody. Bryostatin/Ionomycin (Bryo/Io) provide a useful method of activating tumor-DLNs such that they can readily be expanded to sufficient numbers to be used in AIT, and growing the tumor-DLN lymphocytes in the gamma chain cytokines IL-7 plus IL-15 is superior to IL-2 in terms of T cell numbers and phenotype. AIT with these cells induces tumor regression and provides protection against metastases and future tumor challenge. Here, we provide a stepwise protocol to sensitize tumor-DLN cells in donor mice, activate tumor-DLN T cells ex vivo using Bryo/Io, expansion of these cells in gamma chain cytokines and adoptive transfer of the expanded cells back into tumor-bearing hosts. Methods relevant to these experiments, such as injecting tumor cells intravenously and monitoring for pulmonary metastases, tumor volume measurement and resection, and use of luciferase-expressing tumor cells to monitor for metastases following resection, are described in detail. The methods outlined herein can be easily adapted to suit similar experiments across multiple tumor cell lines and syngeneic mouse models.

Chimeric antigen receptor (CAR) T cells (CAR T) have emerged as a potential therapy for cancer patients. CAR T cells are capable of recognizing membrane proteins on cancer cells which initiates a downstream signaling in T cells that ends in cancer cell death. Continuous antigen exposure over time, activation of inhibitory signaling pathways and/or chronic inflammation can lead to CAR T cell exhaustion. In this context, the design of CARs can have a great impact on the functionality of CAR T cells, on their potency and exhaustion. Here, using CD19CAR as model, we provide a re-challenge protocol where CAR T cells are cultured weekly with malignant lymphoid cell lines BL-41 and Nalm-6 to simulate them with continuous antigen pressure over a four-week period. This protocol can be value for assessing CAR T cell functionality and for the comparison of different CAR constructs.