Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFU,-GM compartment followed by an increase in the number of femoral CFU,. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFU,) compartment expanded to 168% of control, while the more differentiated (CFU,) compartment was reduced (45% of control at day 28). Reductions in both 59Fe-incorporation and erythroblasts/ femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr x 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. The data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.
Preparation of tumour cell supernatantsFourteen-day-old stock LLca tumours were removed aseptically from their hosts. The tumours were weighed and 2.253 g of tumour tissue homogenized in 5 ml of phosphatebuffered saline (PBS) at 4OC. The homogenate was transferred to an iced centrifuge tube and the homogenizing tube washed twice with 5 ml of PBS which was also added to the centrifuge tube. The final volume was centrifuged for 30 min at 2500 rev/min to remove intact cells and cell debris. The supernatant, amounting to 12 ml, was then collected, aliquoted in 3.0 ml fractions, and frozen at -7OOC prior to injection. Each animal received the equivalent of 188 mg of tumour tissue daily to a total tumour weight equivalent of 1880 mg (E a 31-dayold tumour).
Preparation of cell suspensions
Femoral bone-marrow cellsThese were flushed from the excised bone shaft with 3 ml of McCoy's 5A (modified) medium containing 10% heat-inactivated FCS. An aliquot of the cell suspension, following gentle aspiration with a pipet, was counted, with the addition of crystal violet strain containing citrate, with a haemocytometer and a final cell concentration prepared.
Spleen cell suspensionsThese were prepared from excised whole spleens by mincing the tissue in a 1 ml volume of McCoy's 5A (modified) medium and adding an additional 2 ml volume of medium. The