The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared to adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression respectively. This links BRN2 as an activator and conversely MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense ablated cell lines decreased melanoma sphere forming capability, cell adhesion during 3D-spheroid formation, and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than growth as adherent melanoma cells.

In confirmation of Shirai's observation, we find that transplantable mouse tumors grow actively when inoculated into the brains of rats, guinea pigs, and pigeons, whereas subcutaneous or intramuscular grafts in the same animals fail. This growth of foreign tissue in the brain, however, takes place only when the grafted material lies entirely in the brain tissue; if it comes in contact with the ventricle a cellular reaction takes place with resultant destruction of the graft. The growth of foreign tissue in the brain may be completely inhibited by simultaneous inoculations of a small bit of autologous but not by a bit of homologous spleen tissue. Mice highly immune to subcutaneous transplants of mouse cancer show no resistance to such tumors when the inoculation is made into the brain. Although the brain is without obvious power of resistance to implants of transplantable heteroplastic mouse tumors, yet grafts of spontaneous tumors fail to grow there even, as a rule, when tumor implanted and animal host are of the same species.

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It has been a not unusual observation in our experience that mice failing to develop skin cancer as the result of the application of coal tar are found to have tumors of the lungs. The first explanation of the pulmonary tumors which suggests itself is that a cancer of the skin had occurred as a result of the tar, had given off a metastasis to the lung, and had then either healed or sloughed out. But frequent and careful examination of the animals during life failed to show any lesions of the skin suggesting cancer, and at autopsy there were no scars or other evident abnormalities in the painted areas. Furthermore, histological examination of the nodules in the lungs revealed a structure and a type of cell different from the metastases which occur from the tar skin cancers. Spontaneous tumors in the mouse lung have been frequently observed but they rarely occur in such young animals or in such a high proportion of individuals as in our experiments. In the following experiments we have attempted to eliminate completely the probability of skin lesions while determining the influence of the external application of tar on the incidence of primary tumors of the lungs. Experimental Method.It is well known that the induction of skin cancer in mice by the application of coal tar requires frequently repeated applications of this agent. With the product used in this laboratory three applications weekly for 3 months suffice to induce cancer in a fair proportion * This investigation was carried out by means of funds from the Rutherford Donation. 693 on

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Rabbits x-rayed in doses sufficient to reduce the amount of their lymphoid tissue without damage to the bone marrow showed a definite deficiency in the production of precipitins, bacterial agglutinins, and protective antibodies. On the other hand, rabbits subjected to exposures of dry heat sufficient to increase the activity of the lymphoid organs, on immunization develop antibodies in larger quantity than do untreated animals immunized by the same process.