Effect of pregnancy and nursing on the growth of metastases from N-nitroso-N-methylurea-induced mammary carcinomas.

Pregnancy increased the survival times of inbred BUF/N rats bearing occult metastasis of a mammary carcinoma at the time of conception. Nursing following delivery nullified the effect of pregnancy. The similarity of the controlled experimental data with the limited clinical observations is noted. An experimental model of rat mammary carcinoma has been described that possesses a highly metastasizing capacity and can be utilized to study the behavior of clinically silent metastasis.

Expression of kappa-casein in normal and neoplastic rat mammary gland is under the control of prolactin.

An 820-nucleotide-long cDNA clone for the kappa-casein (the casein micelle-stabilizing protein) from rat mammary gland was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence from the nucleotide sequence revealed a signal peptide, 21 amino acids long, and a mature protein of 157 amino acids. The signal peptide of rat kappa-casein was highly homologous to that of the precursor to ovine kappa-casein. However, little homology was apparent when the mature kappa-casein protein sequences from ovine or bovine sources were compared with rat kappa-casein. The kappa-casein mRNA content of the mammary tissue was found to increase during its functional differentiation. Prolactin appears to modulate the production of kappa-casein mRNA. Mammary glands of virgin females had no detectable kappa-casein mRNA; however, a marked induction of kappa-casein mRNA was obtained by intravenous infusion of prolactin. Mammary carcinomas did not follow the same pattern. 7,12-Dimethylbenz[a]anthracene-induced mammary carcinomas had normally low levels of kappa-casein mRNA, but intravenous prolactin infusion increased the levels by 2-fold. The MTW9 mammary carcinoma that grows only in the presence of high levels of mammotropic hormones had kappa-casein mRNA content equivalent to that in 10-day lactating rat mammary gland. Continuous venous infusion of prolactin to MTW9 mammary carcinoma did not modify the kappa-casein mRNA levels. Nitrosomethylurea-induced mammary carcinomas had no detectable kappa-casein mRNA, and intravenous prolactin infusion was unable to induce it.

Temperature gradients and local perfusion in a mammary carcinoma.

Temperature gradients of mammary tumors in randombred Sprague-Dawley rats under normothermia, hypothermia, and hyperthermia were determined, and their experimental modifications were utilized to assess differences in perfusion rates within the neoplastic tissue. Normothermic tumors showed a circadian rhythm with zenith at midnight and nadir at midday. Differences between highest and lowest temperatures recorded during the 24-hour period reached up to 3 degrees C. Similar oscillations were observed in subcutaneous tissue without tumor. An average temperature increment of 0.5-1.0 degrees C was observed when a tumor was transferred from the subcutaneous to the abdominal location. Gradients larger than 1 degrees C were observed within the same tumor in locations only a few millimeters distance from each other. The nonuniformity in temperature within normothermic tumors was exaggerated during hyperthermia. No appreciable change in temperature gradients was seen within a normothermic tumor when tumor blood flow was doubled or reduced to one-third of the basal level. Hyperthermia increased both volume and temperature of tumor efferent blood. As expected, decrease or increase in blood flow during hyperthermia increased or decreased tumor temperature, respectively, but substantial temperature gradients up to 2 degrees C still persisted within adjacent regions. The extent of temperature changes in the tumor could not be correlated with a known change in blood supply. A pulse of cold serum into the tumor afferent artery produced a substantial reduction of tumor blood flow, but only a small depression in tumor temperatures, and a very small change in tumor temperature gradients. No appreciable modification could be brought about in tumor temperature levels and temperature gradients within the tumor by pulses of cold serum in the afferent artery during hyperthermia. After external cooling of the tumor, the time necessary to compensate for temperature depression did not correlate with either the reduction of temperature or with the thickness of the tumor tissue separating the thermistor from the cold source. The results indicate extensive anisotropy of temperature and blood distribution within growing neoplastic tissue and suggest that heat transfer by convection within the tumor is much less effective than it is commonly assumed.

Role of hormones in the growth and regression of human breast cancer cells (MCF-7) transplanted into athymic nude mice.

The hormonal environments require by human breast cancer cells MCF-7 to produce solid tumors in nude mice are described. A 100% take was obtained within 7 days following inoculation of 2X10(6) actively growing (log phase) MCF-7 cells into the mammary fat pads of intact, athymic BALB/c nude mice. Tumors failed to develop, even with an inoculum of 20X10(6) cells/mouse, in ovariectomized mice or in mice made diabetic with streptozotocin and observed for 90 days after cell inoculation. A 100% incidence of tumors was obtained in mice that were either hypophysectomized or made diabetic but received injections of 0.2 IU insulin/day/mouse. A 100% incidence of tumors was also obtained in ovariectomized mice that received 17 beta-estradiol in the form of a pellet placed subcutaneously in the interscapular region at the time of cell inoculation. Palpable tumors also developed in ovariectomized mice treated with prolactin, perphenazine, estrone, or estriol, but no takes were observed in ovariectomized mice treated with progesterone, 5 alpha-dihydrotestosterone, or hydrocortisone. Growth of the MCF-7 tumor was stimulated five- to sixfold in both intact and hypophysectomized mice that each received a 17 beta-estradiol pellet. Removal of the 17 beta-estradiol pellets form tumor-bearing ovariectomized mice failed to induce tumor regression. Tumors that continued to grow in ovariectomized mice deprived of 17 beta-estradiol regressed by 50% or more of their initial volume when tamoxifen was injected for 7 days at 5 micrograms/mouse/day) +/- theophyline (1 mg/mouse/day), tumor growth arrest was observed during the 2-to 3-week treatment period. Streptozotocin-induced diabetes in tumor-bearing mice always resulted in complete tumor regression following a 3-week treatment period.

Fiber optic microfluorimetry for acute and chronic in-vivo animal studies.

Highly localized long-term measurements of tumor transport properties are performed with pairs of adjacent, parallel single-strand optic fibers embedded in a laboratory animal. External illumination and light sensing equipment detect local concentration of a fluorescent indicator initially injected into the venous system. Under appropriate conditions, blood flow per unit volume, capillary permeability, and uptake of fluorescent drugs can be determined from the fluorescence time history. The observation volume has a characteristic dimension on the order of 0.5 mm. Chronic tests in 20 animals monitored for 1-2 weeks have demonstrated that the technique is practical, the tumor histology is acceptable, and the measured local tissue uptake is comparable to average values in the literature. The technique is also suitable for acute experiments using a miniature fiber optic needle probe.

Blood flow and heat transfer in Walker 256 mammary carcinoma.

True thermal conductivity of 13 Walker 256 mammary carcinomas in noninbred Sprague-Dawley rats averaged 3.2+/-0.9 mW/cm/degrees C under physiologic conditions. A comparison of the effective thermal conductivity in 4 tumors with and without blood flow revealed large differences ranging from 14 to 132%. When the blood supply to the tumor was doubled or reduced to one-half, the effective thermal conductivity varied proportionally to the square root of the perfusion rate. The values of thermal conductivity were obtained from a tumor preparation in which blood flow was monitored continuously during temperature changes. These changes were measured by thermistors and produced by thermal probes incorporated by the growing tumor, not surgically inserted within the tissue at the time of measurement. Inasmuch as tissue necrosis was not a dominant factor, the data are interpreted to reflect the degree of difference in local perfusion of the neoplastic tissue.

Bulk transfer of fluid in the interstitial compartment of mammary tumors.

Venous blood leaving a solid tumor showed higher erythrocyte concentration than did aortic blood. Net fluid loss of efferent blood as calculated from hematocrit differences was 2.7 to 6.7% of flow volume, 4.5 to 10.2% of perfusing plasma volume, or 0.14 to 0.22 ml fluid per hr per g in 2 to 5 g transplanted MTW9 and Walker 256 mammary carcinomas, and primary N-nitrosomethylurea- and 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinomas of rats. Net fluid loss was directly related to blood flow but inversely related to tumor size. Increased hydrostatic pressure in tumor interstitial space was a consistent finding. Micropore chambers embedded in transplanted tumors drained 4 to 5 times more interstitial fluid than did identical chambers in the s.c. tissue. It is concluded that: (a) convective currents are present within the interstitial spaces of tumors; (b) the magnitude of fluid transfer can be measured by the difference in hemoconcentration between afferent and efferent tumor blood; and (c) the volume of this fluid transfer is not altered by hormone-induced tumor regression. The increased hydrostatic pressure of tumor interstitial spaces is interpreted as being due to absence of an anatomically well-developed lymphatic network. The bulk transfer of fluid within interstitial spaces is comparable to lymphatic drainage and should be considered in assessing drug concentration and distribution in solid tumors.

Cage for continuous intravascular infusion of isotopes and drugs in small animals.

A cage for continuous intra-arterial or intravenous infusion is described. Major features are: a) The animal is free to move during infusion, b) loss of the infused solution is avoided, c) cleaning and decontamination are simple, and d) infusion can continue for weeks.

N-nitrosomethylurea as mammary gland carcinogen in rats.

N-nitrosomethylurea (NMU) given intravenously to rats at age 50 days induced mammary carcinomas in 89% of BUF/N, 73% of Sprague-Dawley, and 89% of F344 females. Latent periods were, respectively, 77, 86, and 94 days. Mortality was negligible. Biologic properties of NMU-induced tumors were tested in the BUF/N inbred strain. Before treatment, it reduced the number of tumors per rat but not the incidence; and after the tumor was established, castration arrested tumor growth or caused a temporary regression of the tumor. Metastases to bone marrow and spleen were constant, but they were rare to the liver and lungs. After the primary tumor was removed, metastases continued to grow but at a slower rate than the growth of the primary tumor. Almost all tumors were transplantable intraperitoneally and/or subcutaneously in the inguinal area of intact as well as ovariectomized and adrenalectomized rats. Transplanted tumors were able to metastasize as were primary tumors. Doubling times of NMU-induced primary and transplanted carcinomas were similar to 7 days. Cachexia ensued at the 5th week from the onset of the first tumor. When the tumor was larger than 15 g, hypercalcemia was usually observed. The treatment described appears to be the simplest method for inducing in rats a most nearly complete model for human mammary carcinomas.