Pietro M. Gullino

Cell culture on artificial capillaries: an approach to tissue growth in vitro.

Artificial capillaries perfused with culture medium provide a matrix in which cells can attain tissue-like densities in vitro. Products secreted into the medium can be measured as indicators of cell function or may be recovered for other purposes without disturbing the culture.

Angiogenesis as a marker of preneoplastic lesions of the human breast

At the time of biopsy or mastectomy, 947 tissue fragments from 42 patients were transplanted onto the rabbit iris to determine their capacity to elicit new formation of vessels. In all 10 carcinomas tested, at least 1 of the transplanted fragments induced a strong angiogenic response. Of 50 transplants from hyperplastic lobules, 28% also elicited new vessel formation. Fibrous or adipose tissue and tissues from normal lobules, fibrocystic disease, fibroadenoma, lipoma, and gynecomastia almost always failed to produce angiogenesis (Table 1). In the angiogenesis test the human mammary gland behaves like the mouse gland: a fraction of epithelial lesions, which morphologically appear hyperplastic, is already able to induce an angiogenic response just as the carcinomas. In the mouse, the frequency of neoplastic transformation is higher for those hyperplastic lesions with a high frequency of angiogenic response.13 These results suggest that a major effort may be warranted to ascertain whether the frequency of carcinomas is higher in human subjects whose mammary hyperplastic lesions elicit a strong angiogenic response. The angiogenic assay could distinguish lesions undergoing neoplastic transformation before morphological signs of „atypia”︁ and invasion appear.

Natural history of breast cancer: Progression from hyperplasia to neoplasia as predicted by angiogenesis

The age‐adjusted death rate of women with breast cancer has not improved appreciably over the last 20 years. The average duration of life for untreated breast cancer is about three years with a survival rate of about 40% at three years and 18–20% at five years. If the time required for a tumor to double its diameter during a known period of time is taken as a measure of growth rate, one can calculate by extrapolation that two‐thirds of the duration of a breast cancer remains undetectable by the patient or physician. Long before a breast carcinoma can be detected by present technology, metatastic spread may occur and does in most cases. Early removal of the primary lesion is indispensable but not sufficient to prevent metastases. A major task for the biologist is to characterize the preclinical phase of breast cancer, in particular to predict which lesion—morphologically definable only as hyperplastic—already has the potential for unrestrained growth and metastasis. Evidence from our laboratory suggests that progression from hyperplasia to neoplasia can be evaluated by determining whether mammary tissue has acquired the capacity to induce new formation of vessels in the host tissue. The test was done by transplanting tissue fragments onto the iris of rabbits and angiogenesis was evaluated by direct observation through the transparent cornea. Human mammary papillomas and carcinomas, infiltrating or in situ, produced angiogenesis almost constantly, resting mammary tissue almost never, but about 30% of hyperplastic epithelium showed an angiogenic capacity. In the mouse mammary gland, frequency of the angiogenic response increased with the increased frequency of neoplastic transformation of the hyperplastic lesions. In the human mammary gland, the angiogenic capacity of hyperplastic lesions was as frequent as in the mouse gland. Whether the risk of future neoplastic behavior is also similar, should be tested in a prospective clinical trial. A simple method of localizing hyperplastic epithelium can be used for mammary tissue obtained from routine biopsies of “benign” lesions. If the angiogenic property of hyperplastic epithelium can identify a population of women with a mammary parenchyma at higher risk of developing breast carcinoma, a powerful tool is available for predicting neoplastic transformation before clinical manifestations.

Hormone production by cells grown in vitro on artificial capillaries

Abstract An established human choriocarcinoma cell line has been grown within a perfused artificial capillary bed. Cell densities approaching those of tissue have been attained. Cells maintained in this manner produce human chorionic gonadotropin at rates 11 times higher than those grown in conventional monolayer form.

In Vivo Utilization of Oxygen and Glucose by Neoplastic Tissue

The biological role of oxygen utilization and glucose consumption in the survival and growth of tumors was first emphasized by Warburg (1,2). For many years the experimental approach in this field relied upon manometric techniques, tissue slices, and homogenates. The overriding preoccupation of investigators was the aerobic glycolysis as a lesion pathognomonic of neoplastic transformation. Only in 1961 (3) was a tumor preparation developed which permitted measurements of oxygen and glucose consumption by solid tumors in vivo following standard physiological procedures (4–9).

Pharmacokinetics of methotrexate in solid tumors

The transport of methotrexate (MTX) into Walker 256 carcinoma (W256) and hepatoma 5123 (H5123) transplanted in rats was investigated after a pulse injection and continuous infusion of the drug. A mathematical model was developed which adequately described the distribution and transport of MTX in both solid tumors. In H5123 the uptake was limited by the amount of drug carried by plasma (flow-limited transport), but in W256 MTX uptake was limited by the rate at which the drug crossed the tissue barriers (tissue-limited transport). Relative uptake by the solid tumors was almost eightfold more efficient with low than with high doses. MTX concentration in tumor interstitial fluid equilibrated with that of plasma in about 50 hr using a micropore chamber with a diffusion coefficient of 0.5 Μm/min as sampling device. MTX concentration was higher in resistant than in responsive tumors.

Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth

The objective of this chapter is to evaluate the role that neovascularization may have in tumor growth and to assess the present knowledge of tumor angiogenesis. New formation of a vascular network has been recognized to be indispensable for tumor growth since the transplantability of neoplastic tissues was first discovered. Increased knowledge of the angiogenic process is sought for two main purposes: (1) to define the possible relationship between acquisition of angiogenic capacity by a cell population and risk of its neoplastic transformation and (2) to investigate possibilities for effectively influencing angiogenesis and consequently tumor growth. Both questions will be discussed here after the pertinent characteristics of the tumor vascular network have been outlined.

ANALYSIS OF TRANSIENT TEMPERATURE DISTRIBUTIONS IN A PERFUSED MEDIUM DUE TO A SPHERICAL HEAT SOURCE WITH APPLICATION TO HEAT TRANSFER IN TUMORS: HOMOGENEOUS AND INFINITE MEDIUM

Abstract The role of conduction and convection in transferring heat through a perfused medium is quantified by analyzing the transient temperature field within and around a spherical heat source embedded in the medium, using the bio-heat transfer equation. Analytical expressions for transient temperature distributions are obtained in terms of the following dimensionless variables: ξ, the relative position; τ, the Fourier number; β2;, the Peclet number, and ratios of thermal conductivities and difFusivities. The perfused medium is assumed to be infinite, isotropic and homogeneous. Frequency response of the probe to sinusoidal power input is also analyzed. The analytical expressions obtained are used to estimate heat transfer characteristics of tumors from in vivo data describing blood flow and temperature distributions around a spherical probe.

Influence of lithium on mammary tumor growth in vivo.

The possibility that lithium ions stimulate growth of mammary tumors in vivo has been suggested by their mitogenic action in vitro on normal and neoplastic mammary epithelium [8] and their clinical use as stimulators of neutrophil production in tumor-bearing patients treated with cytotoxic drugs [14,15]. Three experiments were performed to assess this possibility. Buffalo/N female rats received a single injection of N-nitrosomethylurea (NMU) at a dose known to produce mammary carcinomas in about 50% of animals under standard conditions. Under lithium treatment, the incidence of tumors did not increase significantly. Sprague-Dawley female rats treated with a single dose of 7,12-dimethylbenz[alpha] anthracene (DMBA), but showing no mammary tumors after 4 months, received lithium in their drinking water for 3 additional months. The number of late-appearing tumors was not increased by lithium treatment. Buffalo/N females with NMU-induced tumors were castrated, and the subsequent changes in tumor volume were compared in lithium-treated and control animals. The regression-regrowth curves were not altered by lithium treatment. These results are in contrast to the growth stimulatory capacity of lithium on mammary epithelium observed in vitro [8] and indicate it is very unlikely that lithium ions have an undesirable growth stimulatory action on primary mammary carcinomas in vivo.

Tissue Culture Models of In Vivo Interferon Production and Action

Interferon participation in recovery from viral infection is clearly documented by a series of in vitro observations. It has been shown in vitro that the inhibition of interferon production during infection often notably increases the amount of virus produced (Glasgow and Habel 1962). Cultures infected with viruses in conditions favoring good interferon production yield scarce quantities of virus and establishment of inapparent or abortive infections (Isaacs, 1963). The same effect can also be obtained by adding preformed interferon to the infected cultures.