In vivo models for numerous diseases and conditions have endpoints that have involved animals being gravely ill or dying. As researchers have sought to utilize animal models in more humane and practical ways, surrogate endpoints have been developed that prevent animals from suffering and provide critical research data. Flow cytometry has been instrumental to these advances. Consider these aspects of preclinical flow cytometry endpoint analysis as you develop new protocols.
Molecular pathology has been used for decades together with anatomic pathology for disease diagnosis and evaluating treatment efficacy. Immunohistochemistry (IHC) has been a key technique used on molecular pathology that involves staining tissue sections with molecular markers and evaluating staining patterns that may correspond to different tumor stages or other disease pathologies.
Non-human primates (NHPs) continue to be a valuable resource for preclinical research because of similarities they share with humans. NHPs, especially rhesus macaques, are used in preclinical studies for evaluating new drugs or vaccines for safety and efficacy. Flow cytometry assays can be easily adapted to study cells from NHP. Consider these three factors if you are planning to adapt a flow cytometry assay for use with NHP samples.
Most preclinical cancer studies are heavily dependent on in vivo (animal) models, especially those using genetically engineered mice. The hundreds of existing mouse models allow scientists to study specific tumor types, including tumors derived from patients (PDX), visualize different immune cell subsets in vivo, and screen large panels of potential drugs or biologics for anti-tumor properties.
The development of new oncology treatments has focused on strategies that alter immune responses. Many of these novel therapies use antibodies that bind to receptors on different immune cell subsets and either activate or suppress their functions depending on the immune response being targeted. Antibody-drug conjugates are also becoming more widely used for improved targeting of drugs to specific cell subsets. Flow cytometry-based receptor occupancy (RO) assays are a valuable tool for quantifying cell surface expression of target molecules and are used to generate pharmacodynamic (PD) data, which can be used with pharmacokinetic (PK) data to guide dosing strategies. Consider these different types of receptor occupancy assays as you develop the most useful tools for screening therapeutic molecules.
Plasmacytoid carcinoma of the urinary bladder – H&E Staining
Immunohistochemistry (IHC) is a technique that originates in the early twentieth century but continues to be a valuable method that forms the backbone of molecular pathology. IHC is used for histological examination of tissues and specifically detects the presence of a molecule, such as a tumor antigen. IHC uses antibody-based labeling in which the primary antibody detects the target of interest and the secondary antibody detects the primary antibody which is linked to a molecule for microscopic visualization. Many different secondary antibody labeling modalities exist, including fluorescence, enzyme-mediated reactions and colloidal gold, and different labels are suited to specific microscopy platforms. Consider these five aspects of IHC as you implement this technique in preclinical cancer research:
The field of oncology has been transformed by the approval and implementation of immune checkpoint inhibitors. Cancer cells can tamper with a variety of self-defense mechanisms, including upregulating expression of immune checkpoint molecules on cytotoxic T cells and natural killer (NK) cells, such as PD-1 and CTLA-4. Overexpression of these markers is associated with T cell exhaustion and allows for tumor cells to evade detection. Immune checkpoint inhibitors target these molecules, which restores immune surveillance and anti-tumor responses by T cells.
Signal transduction networks are essential to normal cell growth and function, but signaling abnormalities, such as those caused by spontaneous mutations in signaling components, are the leading causes of tumor growth. The extracellular signal-regulated kinase (ERK) signaling pathway contributes to normal cell proliferation, differentiation, and survival, but components of the ERK pathway are upregulated in many tumor types, including those associated with melanoma, lung, colon and pancreatic cancer.
DNA damage is one of the primary triggers of cancer development and has been linked to many types of cancers, including prostate, stomach, liver and skin cancers as well as leukemia. Within cells, the DNA sequence encodes all the instructions required for building proteins that are needed for cellular functions such as metabolism, replication, tissue and organ maintenance. The fidelity of the DNA sequence in a cell is maintained by multiple mechanisms but errors and mutations can occur, which sets off a chain of events that lead to tumor growth.
Champions Oncology and others in the field working with patient derived xenografts (PDX) are developing and providing translationally relevant preclinical models to test therapeutic efficacy of IO agents as monotherapy or rational combinations in humanized mice engrafted with functional human immune system.