Mechanistic studies on monocyte migration into multicellular spheroids of breast tumor origin

The majority of invasive-ductal breast tumors develop a desmoplastic reaction. The most abundant stromal host cell type in desmoplastic tumors are fibroblasts. Moreover, invasive ductal breast cancer is characterized by a high MO/MAC content. These immune cells are predominantly located within the fibroblastic stroma surrounding tumor cell islets. The achieved data reveal that 3-D spheroid cultures of both fibroblasts and carcinoma cells from breast tumor origin are infiltrated by MO. Five different breast tumor cell lines (Hs578T, BT549, T47D, MCF-7, BT474) were examined for MO infiltration and CCL2 release. The highly invasive Hs578T cell line showed highest levels of cellular CCL2 production and MO infiltration, while less invasive MCF-7 and BT474 cell lines, expressing very little or no CCL2 were poorly infiltrated. However, the hypothesized correlation between CCL2 expression and MO infiltration was not confirmed by linear regression analysis. To verify the relevance of paracrine factors, particularly CCL2/CCL2R signaling, for MO migration into spheroids of breast tumor origin (especially TAF and highly infiltrated Hs578T breast tumor cells) several experimental series were performed: (1) Disruption of CCL2 gradient by adding recombinant CCL2 to the external milieu of TAF spheroids, almost entirely abrogated the infiltration of TAF spheroids by MO but it does not affect the low, basic migration of MO into normal skin fibroblast spheroids. This result indicates that basic MO migration may relate to mechanisms different from MO infiltration of TAF spheroids. (2) Experiments to block downstream signaling from Gi/o and Gs protein coupled receptors by pertussis toxin (PTX) and cholera toxin (ChTX), respectively were performed. MO recruitment into TAF was inhibited by pre-exposure of MO to PTX and ChTX. However, in contrast to PTX, ChTX could not completely block MO migration. The strong inhibitory effect of PTX is consistent with described in literature involvement of Gi/o proteins in CCL2 signaling, however other 7-transmembrane G protein coupled receptors may also be affected. (3) Therefore, CCL2 receptor signaling was inhibited by pre-exposure of MO to the specific CCR2A/B blocking antibody DOC-3. CCL2/CCR2A/B signaling was shown to be important for MO infiltration into TAF but not Hs578T tumor spheroids This indicates that the migration of MO and also the localization of TAM in tumor tissues are affected by the individual expression pattern of both stromal and tumor cell compartments. A particular high infiltrative blood MO populations according to those described in the literature could not yet be identified. The expression pattern of CD14/CD16 and some antigens relevant for cell-cell interactions (CD49e, CD11a, CD11b) did not indicate the presence of a particular subpopulation. The search was extended to a multigene screening (cDNA array) of migrated versus non-migrated MO. It revealed a number of genes which seem over expressed in migrated cells and which should be validated and further explored in the future. It is concluded that paracrine factors from stromal fibroblasts are relevant to govern and/or at least fine tune the infiltration and location of blood MO in breast tumor tissue where they supposedly undergo abnormal differentiation to become tumor promoting, tumor-associated macrophages. The role of fibroblast-derived CCL2 in this scenario has to be emphasized in light of earlier evidence that the level of tumor derived CCL2 correlates with the abundance of TAM, poor prognosis and clinical outcome in breast tumors