Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

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The overall goal of this preclinical platform is to provide a standard procedure to identify and characterize the mechanisms driving acquired resistance to targeted therapies. This method can help to implement genome drive in oncology, in particular, address key questions about tumor heterogeneity and the multiple mechanisms responsible for acquired resistance to targeted therapy. The main advantage of this technique is the time and cost-effective approach which allows the in-vitro investigation of resistance mechanisms to targeted therapy.

The implication of this technique extend to cancer therapy, as lung resistance is still challenging for emerging targeted therapies. This method can provide insight into the mechanism involved in targeted therapy resistance, but it can also be used to study the efficacy of conventional chemotherapeutic agents. Generally, operators who use this method will stagger with a generation of drug-resistant subclones, the step most difficult to standardize.

We first conceived the idea for this method when we characterized the acquired resistant mechanism to trastuzumab in HER-positive gastric cancer cell lines. Visual demonstration of this method is critical as several techniques are needed for the generation and validation of in-vitro drug resistant models including restoring sensitivity by gene knock-down. To begin this procedure, seed NCI N87 cells into 25 square centimeter culture flasks.

Add 10 micrograms per milliliter of trastuzumab to the culture medium in each flask. Expose the cells to this starting dose until they resume growing. After cell growth has resumed in approximately two weeks, expose cells to a one hundred microgram per milliliter concentration dose of trastuzumab.

Continue gradually increasing the trastuzumab concentration waiting until the cells resume growth before each exposure, until a concentration of 400 micrograms per milliliter is reached. A cell line may exhibit a different sensitivity to different drugs. One solution to this problem is to expose cells to gradually increasing drug doses, starting from a dose about tenfold the hour the plasma peak concentration until the cells keep growing.

Next, seed cells into 24 multi-well culture plates. Expose the seeded cells to increasing target therapy inhibitor concentrations from 100 micrograms per milliliter to 400 micrograms per milliliter. Incubate in a CO2 incubator at 37 degrees Celsius for 15 days.

After this, remove the medium and rinse the cells with 10 milliliters of PBS-1X. Remove the PBS-1X and add between two and three milliliters of fixation solution. After 20 minutes, remove the fixation solution and add two milliliters of 0.5%crystal violet solution to each well.

Incubate at room temperature for two hours. Then, use a pipette to carefully aspirate the crystal violet solution. Immerse the plates in tap water to rinse off any remaining residues.

Air dry at room temperature for up to two days. Next, use an inverted microscope at 4X magnification to count colonies containing more than 50 cells. Calculate the relative resistance IC50 ratio as outlined in the text protocol.

To begin, prepare a single-cell suspension by trypsinization as outlined in the text protocol. Using a hemocytometer and Trypan Blue staining, count the cells. Then, seed 2.5 times 10-to-the-five cells onto a T-25 flask for each condition.

Next, dilute 12.5 microliters of each gene-targeting siRNA oligonucleotide or negative control siRNA oligonucleotide in Minimal Essential Transfection Medium. Add a cationic liposome transfection reagent at a 1:1 ratio with the oligonucleotides. Incubate at room temperature for 20 minutes.

After this, add 700 microliters of the transfection mix to each T-25 flask. Incubate at 37 degrees Celsius for one to three days. Use RT-PCR and Western Blot analysis to verify gene knock-down.

Next, seed cells into 24 multi-well culture plates. Expose the cells to increasing concentrations of target therapy inhibitor from 100 micrograms per milliliter to 400 micrograms per milliliter. For the efficacy of gene knock-down, siRNA oligonucleotides must be specific for reducing target expression.

Designed tools and the measure of siRNA was the same target gene are useful for this purpose. Incubate in a CO2 incubator at 37 degrees Celsius for 15 days. Remove the medium and rinse the cells with 10 milliliters of PBS-1X.

After removing the PBS, add two to three milliliters of fixation solution. Then, remove the fixation solution and add 0.5%crystal violet solution. Incubate at room temperature for two hours.

Using a pipette, carefully aspirate the crystal violet solution. Next, immerse the plates in tap water. Air dry at room temperature for up to two days.

After this, use an inverted microscope at 4X magnification to count the colonies containing more than 50 cells. In this study, three gastric cancer cell lines which express a high level of HER2 and are sensitive to trastuzumab are grown in a culture medium containing the targeted therapeutic agents. From this, three subclones characterized by a stable resistant phenotype are obtained.

Of the subclones, HR NCI N87 is seen to be the most resistant reaching a Relative Resistance IC50 of 28.57. Analysis of protein and gene expression supports the hypothesis that the gene IQGAP1 plays a pivotal role in the resistance phenotype of the HR NCI N87 subline. As shown here, IQGAP1 protein level is fivefold tier in the resistant subclone than in the parental cells.

And IQGAP1 gene expression is 2.5-fold tier in the HR NCI N87 subline than in the original subclone. IQGAP1 gene silencing is then performed to evaluate its role in the development of the trastuzumab resistance phenotype. A clonogenic assay further confirms the hypothesis of IQGAP's importance.

As seen here, the gene silencing restores the cell's sensitivity to trastuzumab, lowering the IC50 value to seven micrograms per milliliter. What I think of this procedure is important to keep in mind that it is not generally easy to obtain drug-resistant cell subclones. And that this method holds a potential failure risk.

Through this procedure it is possible to obtain several cells of clones characterized by different mechanisms driving acquired resistance to target therapies. The development of standard procedure for the in-vitro modeling of target therapy resistance should allow an oncology researcher to investigate novel drug-enabled target to overcome drug resistance which is greatest criticism of the latest generation therapies.

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Journal (Cancer Research)

Journal (Cancer Research)

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

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