Use of Molecular Biomarkers to Quantify the Spatial Distribution of Effects of Anticancer Drugs in Solid Tumors

Abstract

Poor distribution of anticancer drugs within solid tumors may limit their effectiveness. Here, we characterize the distribution within solid tumors of biomarkers of drug effect. g-H2AX, cleaved-caspase-3 or -6, and Ki67 were quantified in tumor sections in relation to blood vessels (recognized by CD31) using monoclonal antibodies and immunohistochemistry. To validate their use, we compared their time-dependent distribution with that of (i) fluorescent doxorubicin and (ii) a monoclonal antibody that detects melphalan-induced DNA adducts. The biomarkers were then used to quantify the distribution of docetaxel in relation to tumor blood vessels. Activation of g-H2AX was evaluated following in vitro exposure of tumor cells to multiple drugs. Distributions of doxorubicin in MDA-MB-231 and MCF-7 xenografts and of melphalan-induced DNA adducts in MCF-7 and EMT-6 tumors decreased with distance from blood vessels, similar to the distributions of (i) g-H2AX at 10 minutes, (ii) cleaved caspase-3 or -6, and (iii) change in Ki67 at 24 hours following treatment. The distribution of these biomarkers following treatment with docetaxel also decreased with increasing distance from tumor blood vessels. Activation of g-H2AX occurred within 1 hour after exposure to several drugs in culture. Multiple anticancer drugs show a decrease in activity with increasing distance from tumor blood vessels; poor drug distribution is an important cause of drug resistance. The above biomarkers may be used in designing strategies to overcome therapeutic resistance by modifying or complementing the limited spatial distribution of drug activity in solid tumors. Mol Cancer Ther; 12(4); 542-52. Ó2013 AACR.

Activity of the hypoxia‐activated pro‐drug TH‐302 in hypoxic and perivascular regions of solid tumors and its potential to enhance therapeutic effects of chemotherapy

“…We have shown previously that gH2aX, cleaved caspase-3 (or -6) and Ki-67 are useful biomarkers to quantify the distribution of drug activity within solid tumors. 15 By using these markers and other methods, our group and others have shown limited distribution of activity of multiple anticancer drugs from blood vessels. 14,15,17,18,24 Thus anticancer drugs are often effective against cells adjacent to vasculature but penetrate poorly through tumor tissue so that they are not delivered in effective concentrations to tumor cells in regions distal to functional blood vessels; moreover, such cells have low rates of cell proliferation and are resistant to cycle-active chemotherapy.…”

“…15,23 TH-302 is a pro-drug that is activated specifically in hypoxic cells, and has demonstrated promising results both in preclinical studies and in early clinical trials. [11][12][13]25,26 However, since TH-302 is non-fluorescent, its direct visualization in tumor tissue is not possible.…”

“…15,23 Briefly, binarized DiOC7 or EF5 images were created and then used to create distance maps. Biomarker images (gH2aX, cleaved caspase-3 or -6 and Ki-67) were used to create binary masks which were then combined with the distance map to form a composite image with distance measurements that corresponded only to the biomarker.…”

“…Expression of gH2aX was evaluated at 10 min after treatment with TH-302 alone or after chemotherapy (given alone or 4 hr after TH-302), while increase in the apoptotic markers and decrease in Ki-67 was evaluated at 24 hr after these treatments; these time points have been shown to give optimal expression of the biomarkers after drug treatment. 15 The distribution of biomarkers after treatment of MCF-7 tumors with doxorubicin, TH-302 and the combination in relation to the nearest patent functional vessel (left panels) and the nearest region of hypoxia (right panels) is shown in Figure 2. In control tumors expression of gH2aX and cleaved caspase-6 was low and Ki67 expression decreased with increasing distance from blood vessels (Fig.…”

“…We have validated techniques to analyze the pharmacodynamic changes that occur after treatment with non-fluorescent drugs by using the biomarkers gH2Ax (a marker of DNA damage), cleaved caspase-3 or -6 (markers of apoptosis) and reduction in Ki-67 (a marker of cell proliferation). 15 Here we quantify the distribution of these biomarkers following treatment of two human xenografts with TH-302 alone or with doxorubicin or docetaxel.…”