The goal of the following ex vivo drug response technique is to implement in the preclinical and clinical development of drugs in an effort to inform on response or IC 50 off target effects and allow for molecular analysis of resistance and feedback mechanisms. This is achieved by first expeditiously procuring a viable tumor specimen that is transported immediately to the laboratory for sectioning. Next, the tissue is embedded in agros, sectioned and treated with a drug of interest at therapeutically relevant doses for various durations.
Finally, the sections are removed from the wells leaving behind any non-responsive tumor outgrowth and the tissue is formula fixed and paraffin embedded before one H and e slide of each treated section is prepared. Results show drug treatment, response of the tumor and or metastasis based on morphological changes of the nuclei indicative of apoptosis. IC 50 determination of drug response, identity of off-target effects and molecular analysis of resistance and feedback mechanisms of non-responsive tumor outgrowth are evaluated.
The main advantage of this technique over existing methods like the evaluation of drug response in established cancer cell lines or cellular isolates from primary patient tumors is that this method allows for the evaluation of drug response in the context of the neighboring microenvironment. It can. It can also help answer key questions in drug development such as tumor selectivity and off-target effects.
That implications of this technique extend toward drug development in the clinical setting when applied, for example, to cornal biopsies obtained from patients enrolled in clinical studies. This technique allows for the analysis of a very small specimen size that is sufficient to require extensive amounts of data. Visual demonstration of this method is critical to address some of the difficulties one may encounter specifically with respect to embedding and sectioning, which can be challenging to learn because not all tumor types are similar and making the appropriate adjustments is key to the success of this technique.
To begin direct the surgical team to deliver the specimen in its fresh state with no formula nor fixative by courier or pneumatic tube system to the pathology department in a tightly sealed and sterile leakproof plastic specimen bag. Within a sterile spill proof container record the harvesting time of the procured specimen, which should be kept rigorously under 30 minutes from the completion of the surgical procedure. Direct the pathology department personnel to use a sterile technique under a laminar flow hood to remove a primary tumor specimen of approximately 0.5 to one cubic centimeter, preferably from the periphery of the index lesion.
Place the specimen in a 15 milliliter sterile conical centrifuge tube containing approximately five milliliters of minimum essential medium or mem containing 1%Antibiotics if available, procure a grossly positive associated lymph node specimen of the same size to compare to the drug response of the primary tumor. If the size of the surgical specimen permits, remove a sample of normal tissue after using mem and antibiotics to transport it to the lab. Transfer the sample to a cryo vial snap, freeze it, and store at negative 80 degrees Celsius.
For future analysis, obtain an optional core needle biopsy or CNB from a clinical study and also transport it to the lab according to the text protocol microwave. A previously prepared 4%agro solution and cool to approximately 25 degrees Celsius. Next, with sterile forceps, place the tissue specimen in an embedding mold and add the liquid aros.
Then position the tissue specimen approximately two millimeters from the cutting surface of the mold to a 24 well plate. Add one milliliter of complete medium to each well and keep at room temperature during the sectioning. For primary breast tumor of invasive ductal carcinoma, use a vibrating microtome.
Set the speed at which the blade approaches the specimen to six and the frequency of the blade to three or these analysis. Set the section thickness to 200 micrometers. Then use a thin layer of liquid adhesive on a specimen disc to mount the specimen, which is eventually submersed in a reservoir of media that is encased in wet ice in preparation for tissue sectioning.
While quickly sectioning to avoid detrimental effects from cold ischemic time, use sterile forceps to immediately remove the precision slice sections and place in a multi-well plate with one milliliter of complete medium. Take multiple control sections that bookend the treated sections to help confirm dose dependent responses. After incubating for one hour to acclimate the samples, add increasing doses of the drug to the sections and further incubate for the treatment period of up to 48 hours or longer depending on the needs.
The experiment following treatment under sterile conditions, remove all sections and transfer to approximately one milliliter of fixative and leave at room temperature overnight after fixation, the specimens are paraffin embedded, sectioned onto charge slides and hematin and eoin stained for evaluation. Shown here. The breast cancer primary tumor and ER positive invasive ductal carcinoma was analyzed ex vivo for treatment response to a heat shock protein 90 inhibitor.
The 2.5 micromolar treatment of the tissue results in a 40%induction of apoptosis as seen by PNO nuclei, whereas an adjacent benign lobule with associated acid eye and duct of the same 2.5 micromolar treated section remains unaltered. This figure of an associated lymph node metastasis also analyzed ex vivo for treatment response to heat shock protein 90 inhibitor demonstrates complete response in loosely associated IDC nests of accompanying lymph node metastasis, unlike tightly formed sphe, which showed no response to treatment and remained viable. Additional analysis of the viable steroids by immunofluorescence revealed a persistent or repression of the STO cell adhesion molecule.
E cadherin as seen here, the ex vivo technique has also been employed on pre-treatment CNBS of clinical studies for correlative analysis, responsive ex vivo sectioned core needle biopsies can be evaluated and later compared to 24 hour post-treatment CMBS for drug retention as measured via liquid chromatography, tandem mass spectrometry analysis and actual patient responds in pet image analysis While attempting the procedure. It is important to remember to work in a quick and efficient manner. After watching this video, you should have a good understanding of how to implement the XB will drug response technique in the preclinical and clinical development of therapeutics as a step towards a personalized medicine approach and therapeutic strategies.
