ABOUT
- Translational medicine focuses on biomarkers in the drug development process and aims to improve the clinical response rate of drug development with precision medicine. It covers from early target confirmation - preclinical R&D - clinical Phase I, II, and III Development, to post-marketing drug testing, through different stages of research to achieve a closed loop of drug development.
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With the continuous development of multi-omics analysis technologies such as genomics, proteomics, and metabolomics, therapeutic methods have expanded from traditional small molecules to technologies such as peptides, proteins, antibodies, gene therapy and cell therapy. However despite recent advances, the causes of many diseases are still not fully understood.Translational medicine transforms biomedical observation and research into interventions to improve health, accelerates the process of basic research, new drug development, and clinical transformation, and becomes an accelerator for precisely targeted therapy.Translational medical research utilizes research methods to determine the relationship between the target and the occurrence and development of the disease. Translational medicine seeks to validate and explore the mechanism of action of the drug, discover biomarkers, develop companion diagnostic products, and identify the most suitable population for clinical research to improve the efficiency and success rate of R&D.
Overlaying translational medicine milestones into drug development[1]
Platforms
Immunohistochemical Technology Platform- Immunohistochemical Technology PlatformImmunohistochemistry, also known as immunocytochemistry, refers to the application of immunology, where antigen detection via an antibody is visualized through a chemical reaction. According to the principle of antigen-antibody reaction and chemical color development, the antigen in the tissue section or cell sample is first bound by the primary antibody. Then an enzyme-linked secondary antibody is used to detect the primary antibodyand DAB is used for color development prior to analysis.Main StepsTissue Processing, Fixation, SectioningAntigen RetrievalRemoval of Endogenous PeroxidaseBlockingPrimary and Secondary Antibody IncubationDetectionCounterstaining
- Tissue Processing, Fixation, SectioningTissue fixation preserves antigens and prevents autolysis and necrosis of harvested tissue. Tissue embedding supports the tissue during slicing, making the slice more solid.
Paraffin Section Frozen Slice Fixed Before embedding: formaldehyde Before or after sectioning: formaldehyde, methanol, ethanol, or acetone Slice Slicer Cryostat Store Store at room temperature for many years One year at -80 °C (longer at -190 °C)长） Advantages Easy to handle without damaging slices ♦ Retains enzyme function and antigenicity
♦ Short protocol (usually does not require lengthy fixation steps)
Limitation ♦ Overfixation can mask epitopes, increasing the need for antigen retrieval.
♦ Long processing time: Gradual dehydration in graded alcohol and xylene to facilitate paraffin penetration.
♦ Without rapid freezing of tissue," ice crystals may form, disrupting tissue structure.
♦ Frozen sections are usually thicker than paraffin sections, which may result in lower resolution and poorer images.
♦ It may be necessary to block endogenous active enzymes.
Paraffin Sectioning vs. Frozen Sectioning - Antigen RetrievalAntigen retrieval is performed on formaldehyde-fixed tissue sections to expose antigenic sites for antibody binding.
Heat-induced epitope retrieval Proteolytic enzyme-induced epitope retrieval Advantages Retrieval of antigenic epitopes is gentler with more controllable parameters. Suitable for antigenic epitopes that are difficult to retrieve. pH value Typically, a pH six buffer is used, but alkaline buffers are also widely used. must be determined experimentally The pH is usually 7.4 Temperature About 95°C. Typically 37°C Incubation time 10-20 minutes 10-15 minutes Buffer Components Depends on the desired pH of the target antigen. Commonly used buffers include sodium citrate, EDTA, and Tris-EDTA Neutral buffer for enzymes such as pepsin, proteinase K, or trypsin Precautions Microwave heating may result in uneven antigen retrieval. Vigorous boiling can cause debonding (separation of tissue from slide). Enzyme repair sometimes disrupts section morphology - concentration and time need to be optimized The Main Method of Antigen Retrieval - BlockingBlocking with serum or BSA prevents nonspecific binding of primary and secondary antibodies to tissues and cells to reduce background and potentially false positive results.❖ Protein blocking: Blocking with serum is critical to prevent nonspecific binding of the antibody to tissue or to Fc receptors (receptors that bind to the antibody constant region (Fc)). Serum from the species of the secondary antibody is a good blocking reagent. Bovine serum albumin (BSA) or casein can be used to block nonspecific antibody binding.❖ Biotin Blocking: When using an avidin/biotin-based detection system, blocks endogenous biotin as it is present in many tissues, especially kidney, spleen, liver, and brain. Incubate the tissue with avidin to block endogenous biotin, followed by incubation with exogenous biotin to block additional biotin binding sites on the avidin molecule.
- Detection❖ Enzyme Chromogenic Method: Chromogenic detection uses enzymes that can catalyze soluble substrates to produce colored precipitates. These enzymes are usually conjugated to secondary antibodies and can also be conjugated to primary antibodies for direct detection.The most commonly used enzymes are HRP, which converts DAB to a brown product, and AP, which converts 3-amino-9-ethyl carbazole (AEC) to a red product. Chromogenic detection is generally more sensitive than fluorescent detection. In addition, unlike fluorescent dyes, colored precipitates are photostable, so stained sections can be preserved for many years.Fluorescence detection requires the use of professional fluorescent microscopes and filters, while chromogenic detection only requires the use of standard microscopes. However, chromogenic assays require more and longer incubation and blocking steps than fluorometric assays.❖ Fluorescence method: Fluorescence detection (immunofluorescence) is based on the characteristic that the fluorophore emits fluorescence with a longer wavelength after being excited by a specific wavelength of light.Fluorescence detection is often used in situations where simultaneous detection of multiple antigens is required. Fluorescent dyes can be conjugated to primary or secondary antibodies or streptavidin.
- Case Studies：PD-L1, Ki-67, Her2, CD31, CD163, FoxP3
IHC Analysis of the expression ofa)PD-L1 from lung adenocarcinoma[3]; b)Ki-67 from periampullary tumors[4]; c)Her2 from lung tumor[5]; d)CD31 from human gastric adenocarcinoma[6]; e)CD163 (M2 TAM marker) from oral squamous cell carcinoma (OSCC)[7]; f)FoxP3 from human glioblastoma[8].
Summary and Outlook- Comprehensive translational medicine platform based on genomics, proteomics, cytomics, and pathomics and high-quality R&D management team; Medicilon translational medicine platform is committed to providing global partners with comprehensive biomarker discovery, target integrated solutions for point-of-care verification, companion diagnostic development, and commercial testing.❖ Protein interaction and protein level biomarker platform constructed by ELISA, ECL (MSD), SIMOA (HD-X), Biacore 8K technology;❖ Cell-level biomarker platform based on flow cytometry (BD Symphony A3, BD Fortesssa, Beckman CytoFLEX S);❖ Multiple nucleic acid-level biomarker platform constructed with fluorescent quantitative PCR technology;❖ Pathological level biomarker platform constructed by immunohistochemistry (TAMs-IHC, FISH) technology.Medicilon is committed to solving the difficulties in developing innovative drugs and the development of precision medicines.
- References：[1] Hugues Dolgos, et al. Translational Medicine Guide transforms drug development processes: the recent Merck experience. Drug Discov Today. 2016 Mar;21(3):517-26. doi: 10.1016/j.drudis.2016.01.003.[2] Ying Xu, et al. A Selective Small-Molecule c-Myc Degrader Potently Regresses Lethal c-Myc Overexpressing Tumors. Adv Sci (Weinh). 2022 Mar;9(8):e2104344. doi: 10.1002/advs.202104344.[3] Jonas J Heymann, et al. PD-L1 expression in non-small cell lung carcinoma: Comparison among cytology, small biopsy, and surgical resection specimens. Cancer Cytopathol. 2017 Dec;125(12):896-907. doi: 10.1002/cncy.21937.[4] Mark M Aloysius, et al. Predictive value of tumor proliferative indices in periampullary cancers: Ki-67, mitotic activity index (MI) and volume corrected mitotic index (M/V) using tissue microarrays. World J Surg. 2010 Sep;34(9):2115-21. doi: 10.1007/s00268-010-0681-3.[5] Montse Verdu, et al. Cross-reactivity of EGFR mutation-specific immunohistochemistry assay in HER2-positive tumors. Appl Immunohistochem Mol Morphol. 2015 Sep;23(8):565-70.[6] Qingling Wang, et al. EPCR promotes MGC803 human gastric cancer cell tumor angiogenesis in vitro through activating ERK1/2 and AKT in a PAR1-dependent manner. Oncol Lett. 2018 Aug;16(2):1565-1570. doi: 10.3892/ol.2018.8869.[7] Faustino J Suárez-Sánchez, et al. Macrophages in Oral Carcinomas: Relationship with Cancer Stem Cell Markers and PD-L1 Expression. Cancers (Basel) (IF: 6.13; Q1). 2020 Jul 2;12(7):1764. doi: 10.3390/cancers12071764.[8] Qi Yue, et al. The prognostic value of Foxp3+ tumor-infiltrating lymphocytes in patients with glioblastoma. J Neurooncol. 2014 Jan;116(2):251-9. doi: 10.1007/s11060-013-1314-0. Epub 2013 Nov 26.[9] Christopher P Austin.Opportunities and challenges in translational science. Clin Transl Sci. 2021 Sep;14(5):1629-1647. doi: 10.1111/cts.13055.
