To reveal the antiangiogenic capability of cancer chemotherapy, we developed an alternative antiangiogenic schedule for administration of cyclophosphamide. We show here that this antiangiogenic schedule avoided drug resistance and eradicated Lewis lung carcinoma and L1210 leukemia, an outcome not possible with the conventional schedule. When Lewis lung carcinoma and EMT-6 breast cancer were made drug resistant before therapy, the antiangiogenic schedule suppressed tumor growth 3-fold more effectively than the conventional schedule. When another angiogenesis inhibitor, TNP-470, was added to the antiangiogenic schedule of cyclophosphamide, drug-resistant Lewis lung carcinomas were eradicated. Each dose of the antiangiogenic schedule of cyclophosphamide induced the apoptosis of endothelial cells within tumors, and endothelial cell apoptosis preceded the apoptosis of drug-resistant tumor cells. This antiangiogenic effect was more pronounced in p53-null mice in which the apoptosis of p53-null endothelial cells induced by cyclophosphamide was so vigorous that drug-resistant tumors comprising 4.5% of body weight were eradicated. Thus, by using a dosing schedule of cyclophosphamide that provided more sustained apoptosis of endothelial cells within the vascular bed of a tumor, we show that a chemotherapeutic agent can more effectively control tumor growth in mice, regardless of whether the tumor cells are drug resistant.

https://cancerres.aacrjournals.org/content/canres/60/7/1878.full.pdf

Antiangiogenic Scheduling of Chemotherapy Improves Efficacy against Experimental Drug-resistant Cancer

Three major models (from Tofts, Larsson, and Brix) for collecting and analyzing dynamic MRI gadolinium-diethylene-triamine penta-acetic acid (Gd-DTPA) data are examined. All models use compartments representing the blood plasma and the abnormal extravascular extracellular space (EES), and they are intercompatible. All measure combinations of three parameters; (1) kPSp is the influx volume transfer constant (min-1), or permeability surface area product per unit volume of tissue, between plasma and EES; (2) ve is the volume of EES space per unit volume of tissue (0 < ve < 1); and (3) K(ep), the efflux rate constant (min-1), is the ratio of the first two parameters (k(ep) = kPSp/ve). The ratio K(ep) is the simplest to measure, requiring only signal linearity with Gd tracer concentration or, alternatively, a measurement of T1 before injection of Gd (T10). To measure the physiologic parameters kPSp and ve separately requires knowledge of T10 and of the tissue relaxivity R1 (approximately in vitro value).

Modeling tracer kinetics in dynamic Gd-DTPA MR imaging

Chelation of the rare-earth element gadolinium (Gd) with diethylenetriaminepentaacetic acid (DTPA) results in a strongly paramagnetic, stable complex that is well tolerated in animals. The strongly paramagnetic gadolinium complex reduces hydrogen-proton relaxation times even in low concentrations (less than 0.01 mmol/L). The pharmacokinetic behavior of intravenously delivered Gd-DTPA is similar to the well known iodinated contrast agents used in urography and angiography; excretion is predominantly through the kidneys with greater than 90% recovery in 24 hr. The intravenous LD50 of the meglumine salt of Gd-DTPA is 10 mmol/kg for the rat; in vivo there is no evidence of dissociation of the gadolinium ion from the DTPA ligand. The combination of strong proton relaxation, in-vivo stability, rapid urinary excretion, and high tolerance favors the further development and the potential clinical application of gadolinium-DTPA as a contrast enhancer in magnetic resonance imaging.

https://www.ajronline.org/doi/pdfplus/10.2214/ajr.142.3.619

Characteristics of gadolinium-DTPA complex: a potential NMR contrast agent.

Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures

Abnormal choline metabolism is emerging as a metabolic hallmark that is associated with oncogenesis and tumour progression. Following transformation, the modulation of enzymes that control anabolic and catabolic pathways causes increased levels of choline-containing precursors and breakdown products of membrane phospholipids. These increased levels are associated with proliferation, and recent studies emphasize the complex reciprocal interactions between oncogenic signalling and choline metabolism. Because choline-containing compounds are detected by non-invasive magnetic resonance spectroscopy (MRS), increased levels of these compounds provide a non-invasive biomarker of transformation, staging and response to therapy. Furthermore, enzymes of choline metabolism, such as choline kinase, present novel targets for image-guided cancer therapy.

/pdf/choline-metabolism-in-malignant-transformation-4s9se90sd8.pdf

Choline metabolism in malignant transformation

NMR study of in vivo RIF-1 tumors: Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

In vitro labeling and gold activation autoradiography were used to determine the [3H]thymidine ([3H]TdR)-labeling indices and DNA synthesis times for C3H/He spontaneous mammary tumors. Three variations of the [3H]TdR, [14C]thymidine ([14C]TdR) double-labeling method, together with double-emulsion autoradiography, were used to determine the DNA synthesis times (TS). Tumors labeled totally in vivo (in vivo-in vivo method) and tumors labeled with [3H]TdR in vivo and subsequently labeled with [14C]TdR in vitro showed similar TS values. DNA synthesis times for tumors determined totally in vitro by double labeling (in vitro-in vitro method) were significantly longer than those observed in vivo; however, identical samples subjected to Hypaque-Ficoll gradient separation after double labeling showed TS's similar to those found in vivo. Furthermore, the interval between [3H]TdR and [14C]TdR administration had no effect on TS estimates in vitro. Gold activation autoradiography was used in the present experiments to reduce autoradiographic exposure times. This method, together with in vitro labeling, permits [3H]TdR labeling index and TS determinations after 6-hr and 7-day exposures, respectively.

In vitro labeling and gold activation autoradiography for determination of labeling index and DNA synthesis times of solid tumors.

The tumor growth fraction measured by the percentage labeled mitoses method has been determined in transplantable solid and ascites murine tumors, the latter being measured at different times after transplantation. These values were compared to an in vitro , autoradiographic assay that determines the fraction of cells in a given population (primer-available DNA-dependent DNA polymerase index) that have both nuclear DNA-dependent DNA polymerase and DNA capable of acting as primer-template. It appears that almost all cells with a short G1 phase duration (<19 hr) that are within the proliferative cycle are primer-available DNA-dependent DNA polymerase positive. The results of the comparison indicate that the primer-available DNA-dependent DNA polymerase index estimation of growth fraction is very nearly identical to the growth fraction measured by the percentage labeled mitoses method.

https://cancerres.aacrjournals.org/content/canres/36/7_Part_1/2415.full.pdf

Estimation of tumor growth fraction in murine tumors by the primer-available DNA-dependent DNA polymerase assay.

The cellular kinetics of human mammary tumors were studied by in vitro methods. These techniques include single 3HTdR labeling to measure the 3HTdR LI, double labeling with 3HTdR and 14CTdR to measure DNA synthesis time, and an estimation of the growth fraction by the PDP index. Calculations of the potential doubling time and cell cycle time were made from these measurements. The 3HTdR LI of primary malignant tumors was greater than that of benign tumors, but only half that of metastatic lesions. There was considerable heterogeneity in the 3HTdR LI of primary tumors, but the DNA synthesis times were relatively invariant. Estimation of the growth fraction by the PDP index also revealed extensive heterogeneity, but the primary tumors were not different from metastases. There appear to be subsets of tumors with high and low proliferative values that correlate with some clinical parameters, such as age and nodal positivity. This material provides a data base for stratification of patients for future protocols and the use of cell kinetics in treatment programs.

The cell kinetics of human mammary cancers

The present studies were conducted to assess the antiproliferative effects of dexamethasone (DEX) in murine, rat, and xenograft tumor models and to determine if this kinetic response could be correlated with the level of cytosolic glucocorticoid receptors. Saturable DEX receptors were determined by the dextran-coated charcoal competitive-binding assay, and the antiproliferative effects of DEX were determined by serial measurements of [3H]thymidine labeling indices before and after DEX treatments. The results from such studies in 3 colon tumor lines, one mouse (CO-38) and 2 human xenograft lines (LoVo, H81-4), were qualitatively similar. [3H]Thymidine labeling indices, initially reduced 50 to 60% by the DEX treatments, subsequently increased to as much as 2 times control values, 24 to 36 hr later. With the R3230AC rat mammary tumor model, the DEX dose-dependent antiproliferative effect was similar regardless of whether the tumor was grown in its syngeneic host, Fischer 344 rats, or as a xenograft in athymic nude mice. Studies using the B16 melanoma and SaD2 sarcoma tumor models indicated that the in vivo efficacy of vincristine, given after DEX, was highly sequence dependent. The results from these and other studies in glucocorticoid receptor-positive solid tumor models indicated that the DEX dose-dependent antiproliferative effect and the timing for maximal post-DEX [3H]thymidine labeling indices can be directly correlated with the level of saturable glucocorticoid receptors.

https://cancerres.aacrjournals.org/content/canres/43/10/4757.full.pdf

Lewis M. Schiffer

Papers

NMR study of in vivo RIF-1 tumors: Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

In vitro labeling and gold activation autoradiography for determination of labeling index and DNA synthesis times of solid tumors.

Estimation of tumor growth fraction in murine tumors by the primer-available DNA-dependent DNA polymerase assay.

The cell kinetics of human mammary cancers

Correlation between glucocorticoid receptor content and the antiproliferative effect of dexamethasone in experimental solid tumors.