Two variants (A and B) of the widely employed Walker 256 rat tumor cells are known. When inoculated sc, the A variant produces solid, invasive, highly metastasizing tumors that cause severe systemic effects and death. We have obtained a regressive variant (AR) whose sc growth is slower, resulting in 70-80% regression followed by development of immunity against A and AR variants. Simultaneously with the beginning of tumor regression, a temporary anemia developed (~8 days duration), accompanied by marked splenomegaly (~300%) and changes in red blood cell osmotic fragility, with mean corpuscular fragility increasing from 4.1 to 6.5 g/l NaCl. The possibility was raised that plasma factors associated with the immune response induced these changes. In the present study, we identify and compare the osmotic fragility increasing activity of plasma fractions obtained from A and AR tumor bearers at different stages of tumor development. The results showed that by day 4 compounds precipitating in 60% (NH 4 ) 2 SO 4 and able to increase red blood cell osmotic fragility appeared in the plasma of A and AR tumor bearers. Later, these compounds disappeared from the plasma of A tumor bearers but slightly increased in the plasma of AR tumor bearers. Furthermore, by day 10, compounds precipitating between 60 and 80% (NH 4 ) 2 SO 4 and with similar effects appeared only in plasma of AR tumor bearers. The salt solubility, production kinetics and hemolytic activity of these compounds resemble those of the immunoglobulins. This, together with their preferential increase in rats bearing the AR variant, suggest their association with an immune response against this tumor.

Cancer anemia is classified as an anemia of chronic diseases, although it is sometimes the first symptom of cancer. Cancer anemia includes a hemolytic component, important in the terminal stage when even transfused cells are rapidly destroyed. The presence of a chronic component and the terminal complications of the illness limit studies of the hemolytic component. A multifocal model of tumor growth was used here to simulate the terminal metastatic dissemination stage (several simultaneous inoculations of Walker 256 cells). The hemolytic component of anemia began 3-4 days after inoculation in 100% of the rats and progressed rapidly thereafter: Hb levels dropped from 14.9 ± 0.02 to 8.7 ± 0.06 from days 7 to 11 (~5 times the physiologically normal rate in rats) in the absence of bleeding. The development of anemia was correlated (r 2 = 0.86) with the development of other systemic effects such as anorexia. There was a significant decrease in the osmotic fragility of circulating erythrocytes: the NaCl concentration causing 50% lysis was reduced from 4.52 ± 0.06 to 4.10 ± 0.01 (P<0.01) on day 7, indicating a reduction in erythrocyte volume. However, with mild metabolic stress (4-h incubation at 37 o C), the erythrocytes showed a greater increase in osmotic fragility than the controls, suggesting marked alteration of erythrocyte homeostasis. These effects may be due to primary plasma membrane alterations (transport and/or permeability) and/or may be secondary to metabolic changes. This multifocal model is adequate for studying the hemolytic component of cancer anemia since it is rapid, highly reproducible and causes minimal animal suffering.

Cancer pathognomonic systemic effects (PSE) have high individual variability. For this reason present data were collected daily and synchronized considering four main points: inoculation day, onset of PSE, aggravation and death. The subclinical period free of PSE ranged between 15.7 +/- 2.2 days, the clinical period was less variable, 8.9 +/- 0.5 days, divided in a moderate and a grave phase of nearly the same length. PSE involved disturbances of fundamental homeostatic regulations: appetite, sodium, water, immune, etc. PSE triggering correlated highly with survival (r2 = 0.95, P < 0.01), but poorly with primary tumor growth, and it was anticipated by metastases from 20.5 +/- 2.6 to 10.6 +/- 1.1 days (P < 0.01). After multifocal simultaneous inoculations, PSE triggering was anticipated to 4.2 +/- 0.2 days (marked reduction of individual variability), in the presence of small total-tumor masses, absence of macroscopic metastases, and without changes in the following clinical period features. PSE triggering seems to be a major prognostic indicator probably related to multifocal tumor growth.

In the course of studies on the effects of septal area lesions on neuroimmunomodulation and Walker 256 tumor development, it was observed that tumor-induced sodium and water retention was less marked in lesioned than in non-lesioned rats. In the present study possible mechanisms involved in this phenomenon were investigated. The experiments were performed in septal-lesioned (LW; N = 15) and sham-operated (SW; N = 7) 8-week-old male Wistar rats, which received multifocal simultaneous subcutaneous (sc) inoculations of Walker 256 tumor cells about 30 days after the stereotaxic surgery. Control groups (no tumor, sham-operated food-restricted (SFR), N = 7) and lesioned food-restricted (LFR, N = 10) were subjected to a feeding pattern similar to that observed in tumor-bearing animals. Multifocal inoculation of Walker 256 tumor rapidly induces anorexia, which is paradoxically accompanied by an increase in body weight, as a result of renal Na + and fluid retention. These effects of the tumor were also seen in LW rats, although the rise in fractional sodium balance during the early clinical period was significantly smaller than in SW rats (day 4: SW = 47.6 ± 6.4% and LW = 13.8 ± 5.2%; day 5: SW = 57.5 ± 3.5% and LW = 25.7 ± 4.8%; day 6: SW = 54.4 ± 3.8% and LW = 32.1 ± 4.4%; P<0.05), suggesting a temporary reduction in tumor-induced sodium retention. In contrast, urine output was significantly reduced in SW rats and increased in LW rats and SW up to 4.5 ml/100 g body weight), with no change in osmolar excretion. These temporary changes in the tumors effects on LW rats may reflect a reversal of the secondary central antidiuretic response induced by the tumor (from antidiuretic to diuretic).

Sodium retention is a frequent effect of cancer in humans and animals, but the mechanism involved is not yet understood. In the Walker-256 tumor, sodium retention has been considered to be a late effect, secondary to retention in the tumor mass, and/or to adrenal hypertrophy. Normally, (in rats receiving single tumor implants), the development of different tumor systemic effects (TSE) such as anorexia, sodium and fluid retention, anemia and immune depression in rats is synchronous within each individual but random among individuals of a given group in which they appear 6-47 days, or more, after inoculation. In present study, multifocal simultaneous inoculations of tumor cells resulted in a rapid and synchronous initiation of TSE (in 3-4 days) in all rats when no local effects of metastases could mask the results. Sodium retention is a special tumor effect on Na+ balance and a very sensitive indicator of TSE initiation. The results from multifocally inoculated rats were averaged in each (sub-clinical (SubC), moderate (mCP) and grave (gCP)) clinical phase and compared to food-restricted (FR) rats. There was a significant, early decrease in urinary Na+ excretion during mCP when compared to SubC and FR. The renal sites involved were studied in awake, unrestrained animals by measuring of sodium, creatinine and lithium clearances. There was an initial increase in the absolute proximal (mCP: 21.4 +/- 1.7 vs FR: 16.0 +/- 1.1 mmol/min/100 g b.w., p < 0.05) and post-proximal (mCP: 11.1 +/- 0.4 vs FR: 6.6 +/- 0.4 mmol/min/100 g b.w., p < 0.001) Na+ reabsorption, which were partially compensated for by a rise in glomerular filtration rate (mCP: 213 +/- 11.4 vs FR: 162 +/- 10.2 microL/min/100 g b.w., p < 0.01) and by a fall in fractional proximal Na+ reabsorption (mCP: 62.8 +/- 2.2% vs FR: 70.1 +/- 1.7%, p < 0.05), despite significant Na+ and fluid retention. The terminal phase of illness (gCP) culminated with a marked decrease in creatinine clearance, suggesting a significant fall in renal function. The multifocal model proved useful for studying the initial TSE, since the sites of action would, in principle, be easy to identify. These observations may be of physiological interest since TSE may result from the abnormal production of physiological modulators.