To investigate this question in vivo Robert Weinberg and colleagues subcutaneously injected two different populations of tumour-forming human mammary epithelial cells into opposite flanks of a nude mouse. “Instigator cells” can form invasive ductal adenocarcinoma-like tumours and recruit stromal cells, whereas “responder cells” inefficiently generate tumours (slow-growing tumours arise in ∼25% of injected mice). However, when instigator cells were grown in the opposite flank the responder cells formed tumours in 100% of the mice and these were approximately 10-fold larger. The responder tumours were not infiltrated by metastases from the instigator tumours, and the growth of the responder tumours paralleled the growth of the instigator tumours, indicating that the instigators generate a supportive systemic environment for tumour growth.
Conditioned media from cultured instigator cells did not increase the growth of the responder cells in vitro, suggesting that responders are not directly affected by instigator-mediated endocrine signalling. So do instigators affect the recruitment of BMDCs? The authors transplanted green fluorescent protein (GFP)-expressing bone marrow cells into immunocompromised mice and found that their accumulation in responder tumours was significantly increased when grown opposite instigator tumours. Furthermore, they mixed unfractionated bone marrow cells from instigator-bearing mice and responder cells prior to injection into mice and found that the size and incidence of responder tumours increased. These data indicate that endocrine perturbation of host bone marrow by the instigator tumours mobilizes bone marrow cells, promoting tumour growth. Next, they assessed which cells in the bone marrow are affected and found that the Lin−SCA1+KIT+ (LSK) compartment, which contains haematopoietic stem cells, was significantly decreased in the bone marrow. So they measured the expression of SCA1 and KIT on responder-associated GFP-BMDCs and found that most SCA1+ cells were KIT−, corresponding to quiescent haematopoietic progenitor cells. Therefore, instigation appears to decrease the haematopoietic stem cell compartment in the bone marrow, which correlates with the accumulation of quiescent progenitors in the responding tumours. How do instigators mediate these effects? Out of 80 cytokines analysed, only osteopontin levels were increased in the plasma from mice in which instigators were grown. Osteopontin restricts the levels of LSK cells in the bone marrow and is a clinical marker of poor prognosis for patients with metastatic breast cancer. When the secretion of osteopontin was reduced in instigator cells through short hairpin RNA, growth of the responders was no longer stimulated; however, a population of non-instigating tumour cells forced to express osteopontin did not instigate the growth of contralaterally injected responder cells, indicating that osteopontin is necessary but not sufficient for instigation.
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