A molecular case report: functional assay of tyrosine kinase inhibitors in cells from a patient's primary renal cell carcinoma

Abstract

Current therapies for Renal Cell Carcinoma favor vascular endothelial growth factor receptor (VEGF-R) tyrosine kinase (TK) inhibitors (TKIs). In theory, these are most applicable in tumors that have lost VHL-with subsequent stabilization of HIF and upregulation of VEGF. A subset of patients harbor primary-refractory disease, as in this case, where there was no evidence for loss of VHL or chromosome 3p. We evaluated molecular targeted agents in viable tumor cells cultured from a patient's clear cell renal cell carcinoma (RCC). Of 66 agents, only dasatinib, an inhibitor of Src tyrosine kinase, strongly reduced viability of the patient's cultured kidney tumor cells. Immunostaining of the original primary tumor revealed strong positivity for VHL and Src protein expression. Functional evaluation of a patient's tumor cells appears feasible in the setting of RCC.

Keywords: molecular targeted therapy; renal cell carcinoma; tyrosine kinase.

Figure 1. Functional assays of Ki-CA patient tumor derived cells’ response to molecular targeted small-molecule kinase inhibitors. Two thousand cells in medium without EGF were added to 96-well plates containing each small-molecule inhibitor at four serial dilutions spanning a concentration range that includes the predicted IC₅₀, incubated at 37°C for three days and assayed by MTS (Promega). The viability data were adjusted for wells with no cells/inhibitor, and normalized to untreated wells of the cultured cells (OD value for wells with cells without drug treatment = 100% cell viability) and to a database of cell lines and tumor samples to yield responses and the IC₅₀ in nM for each agent. IC₅₀ values less than 20% of the global median (shown as 100%) are considered responses and are well within clinically achievable concentrations.

Figure 2. Dose responses of Ki-CA patient derived cell line to selected drugs. Cells were added to 96-well plates containing each small-molecule inhibitor at 11 serial dilutions spanning a concentration range that includes the predicted IC₅₀ and evaluated as in Figure 1. Values shown are mean ± SD for triplicate wells. Best fit curves were generated using GraphPad Prism software.

Figure 3. Primary Ki-CA patient tumor immunohistochemistry. Tumor exhibited both Src positivity (A) and VHL positivity (B). Power: 20 × ; Scale bar: 20 μM