In-Cell Detection By Flow Cytometry of JAK2 V617F Mutation Using Gold Nanoflares

Today's conventional technology has to overcome many challenging aspects for integrating expressional analysis of a mutation into the cellular function of a living cell. It is very striking to detect the expressional status of a mutation in a living cell without compromising its cellular function as well as its viability that allows to perform subsequent functional analysis. This has been a long time goal for most of the biological studies.

Myeloproliferative neoplasms (MPN) originate at the level of hematopoietic stem cell with a single nucleotide mutation in the cytosolic tyrosine kinase, Janus kinase 2 (JAK2) named as JAK2V617F which facilitates the constitutive intracellular activation of JAK2 and found in 95% of Polycythemia Vera (PV) patients. Detection of this mutation is a major criteria for diagnosis of the malignancy and changes in the expression levels of JAK2V617F is valued for the prognosis as well as response to the treatment.

Here, we have utilized the nanotechnology's new development, entitled as "multiplexed nanoflares", a gold nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are highly oriented oligonucleotide sequences, which are hybridized to a reporter dye such as Cy3 and Cy5 and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra and intracellular environments. Thus, this signal could be easily detected by flow cytometry or confocal microscopy.

We have employed JAK2 V617F specific nanoflare probes labeled with Cy3 (JAK2V617F-Cy3) and JAK2 wild type (wtJAK2) nanoflare probe labeled with Cy5 (JAK2-Cy5) including negative and positive control probes to distinguish and isolate mRNA's of JAK2V617F and wtJAK2 in live cells by measuring subsequent fluorescence with flow cytometry. The initial testing of the nanoflares has been performed by treating cell lines such as K562 (wtJAK2), SET-2 (JAK2V617F heterozygote), HEL (JAK2V617F homozygote) cells with nanoflare probes which did not alter cell viability and morphology with the applied concentrations during 18 hours of cell culture. We measured the levels of two mRNA targets, JAK2V617F and JAK2, and detected 90±6 % positivity with JAK2V617F-Cy3 treated HEL cells and no positivity in K562 cells, meanwhile wtJAK2 probe treated cells succeeded staining of SET and K562 cells. Real time reverse transcription PCR (RT-PCR) was performed on the same cells to compare the relative amounts of target RNA in each cell type by calculating relative changes in mRNA expression using the ΔΔCt (cycle threshold) method and comfirmed the results of flow cytometry with sensitivity of 0.001. Subsequently, we have tested mononuclear cells (MNC) received from peripheral blood of 5 patients with PV, one of which has JAK2V617F heterozygote allele (patient 5) and 4 patients had JAK2V617F homozygote allele (patients 1-4)which were tested with nested allel specific PCR. Flow cytomeyric analysis of the patients` MNC treated with the wtJAK2 and JAK2V617F probes demonstrated distinct populations of wtJAK2⁺ and JAK2V617F⁺. Populations of wtJAK2⁺/JAK2V617F⁺ (2.9%), wtJAK2^-/JAK2V617F^- ( 21.4%), wtJAK2⁺/JAK2V617F^-( 0%), wtJAK2^-/JAK2V617F⁺ ( 79%) of patient 5 and populations of JAK2V617F⁺ (36,8%± 5 ), JAK2V617F^- of patients 1-4 were sorted by cell sorter with ~ 90% purity following detection and analysis of each populations for relative changes in mRNA expressions . The data revealed distinct ΔCt values between each populations.

In this study, we have aimed for a new approach by utilizing nanotechnology aligned with the conventional techniques in order to save time, money and outcome. The capability of in-cell detection and isolation of the JAK2V617F and wtJAK2 first time by performing the expression analysis, changes the routine protocols backwards, enables the detecting direct effect of the mutation to the cells as well as other genes. This study creates a platform where functional analysis regarding to the more detailed understanding of the initiation of malignancy as well as responses to the therapies could be performed.