7thAnnual NIH SBIR/STTR Conference
Drug Response Indicators in Cancer Cells Provide the Basis of Personalized Anticancer Chemotherapy
Personalized anticancer chemotherapy based on measurement of drug response indicators (DRI) expressed by cancer cells. Ts'o, P.O.P.1, Lesko, S.A.1, Lum, Z.P.1 , Deamond, S.F.1, Shan, E.1, Zhou, D.B.1, Daniel, J.R.1. 1 CCC Diagnostics, LLC, Baltimore, MD.
Targeted therapy in cancer patients coupled with the diagnostic tools to measure the target status in individual patients form the basis of personalized anticancer chemotherapy (PAC). The targeted therapies for cancer are designed against specific cellular components (receptors, enzymes) or processes (repair, mitotic division). The targets, termed drug response indicators (DRI) can now be evaluated by immunochemical staining since fluorescent dye labeled monoclonal antibodies (MAB) are available to stain these cellular targets. Quantitative numbers can be derived from fluorescence microscopy measurements and normalized to a reference standard for inter-laboratory comparison. For interpretation of the fluorescence data, particularly for the establishment of a cut off level distinguishing the responsive tumor cells from the resistant tumor cells, we utilized a battery of cancer cell lines with different sensitivities to various anticancer drugs. The inhibition of in-vitro cell growth by each drug was correlated to expression of the related DRI. This report describes an in-vitro experimental system for generating drug response data from breast cancer tumor cells, and correlating it with the level of expression of respective DRI.
Methods: Linear standard curves of fluorescence intensity vs. exposure time were generated from digital images for each microscope filter cube utilizing 6 um fluorescent microspheres. Human breast cancer cell lines, fixed on microscope slides, were incubated simultaneously with 3 different MAB (Herceptin, anti-estrogen receptor and anti-beta tubulin III) labeled with different fluorescent dyes, plus anti-pancytokeratin (CK) for epithelial cell visualization. Digital images of the same field of cells were acquired for each of the 4 fluorescent markers using exposure times that produce a fluorescence of 2000 with standard microspheres as reference. The spatial area of the cells, outlined using CK fluorescence, was overlaid on an identical field of cells in a DRI image in order to quantify DRI expression. The effect of DRI related drugs (Tamoxifen, Paclitaxel, Herceptin) on the proliferation of human cancer cell lines in culture was determined spectrophotometrically by measuring the bioreduction of a tetrazolium salt.
Results: The cytotoxic response of various breast cancer cell lines to Herceptin correlated inversely with the level of HER-2/neu expression (Herceptin binding), evidenced by a Spearman rank coefficient of (-1). There was an inverse correlation between the cytotoxic response of breast cancer cell lines treated with Tamoxifen and level of estrogen receptor expression (specific MAB), shown by a Spearman rank correlation coefficient of (-.995). The cytotoxic response of various breast cancer cells to paclitaxel corresponds with expression of beta-tubulin III. (Spearman rank correlation coefficient of +.995). Cell lines displaying a lower level of binding beta-tubulin III were sensitive to treatment with paclitaxel. In contrast, cell lines displaying a higher level of binding beta-tubulin III displayed a slight response to higher doses of paclitaxel. Measurement of DRI in the circulating cancer cells in breast cancer patients is currently in progress.
Paul OP Ts'o
CCC Diagnostics LLC
Caton Research Center
3918 Vero Rd
Suite B
Baltimore, MD 21227
(410) 633-4885
(410) 633-4502 Facsimile