Samenvatting
Macrophages are often found to be present in tumors in high numbers; and this is correlated with a shorter survival of the patients. Tumor-associated macrophages (TAM) are associated with an M2 "alternatively activated" profile (CD206+, CD163+) and function as promoters of tumor growth by inducing angiogenesis, invasion and metastasis; protecting tumor cells from therapy-induced apoptosis; and suppressing the immune system mainly in hypoxic regions. Macrophages with an M1 "classically activated" profile on the contrary stimulate immune responses in normoxic regions. The aim of our study was to investigate the characteristics of macrophages in multiple myeloma (MM) looking at phenotype and drug resistance.
We identified TAM on bone marrow sections of MM patients by immunostainings for CD163. A higher number of CD163+ cells was noticed in the MM samples compared to normal samples. Further analysis of the number CD163+cells in healthy areas and MM infiltrated areas confirmed that the higher amount of CD163+ originates from MM infiltrated areas.
In addition, in the 5T33MM mouse model the phenotype of bone marrow located macrophages was investigated. Macrophages isolated from both 5T33MM and naive mice revealed a different expression of several macrophage surface markers: a higher expression of CD206, CD80 and CD115; a lower expression of CD86, CCR7, Ly6G and MHCII on the membrane of 5T33MM macrophages compared to naive macrophages. According to these data, macrophages isolated from 5T33MM mice had more frequently an M2 profile (CD206+) compared to macrophages isolated from naive mice (pNext, we generated TAMs in vitro. CD11b+ cells, isolated from naive mice, were incubated with conditioned medium (CM) from 5T33MMvitro cells. After 1 week culture, the phenotype of surviving cells was analyzed. The bulk population was positive for the macrophage marker F4/80. Also a higher expression of CD206 was observed on generated TAMs compared to the starting population. 5T33MMvitro CM contains Interleukin-10 (IL-10) which is described as a M2 macrophage differentiator. Addition of IL-10 blocking antibodies reduced CD206 upregulation on generated TAMs with an average of 30% compared to control.
The involvement of TAMs in drug resistance was investigated. In vitro generated TAMs were cocultured with 5T33MMvt cells with or without bortezomib or melphalan. TAMs increased the survival of 5T33MMvt cells compared to control (pPreliminary results performed with ex vivo isolated macrophages from 5T33MM mice, also demonstrated a protection from apoptosis of 5T33MMvt cells when co-cultured with ex vivo isolated macrophages. This protective effect remains when bortezomib 5-10nM or Melphalan 15µM were added.
In conclusion, we analyzed the presence and characteristics of macrophages in MM. The number of macrophages is higher in bone marrow samples from MM patients compared to healthy persons. Also, a higher percentage of M2 macrophages (CD11b+/F4/80+/CD206+) was observed in the bone marrow of 5T33MM mice compared to naive mice. In vitro generation of M2 macrophages with 5T33MMvt CM is at least partially IL-10 mediated. Furthermore, MM-associated macrophages contribute to a better survival of 5T33MMvt cells and protect them against several anti-myeloma drugs.
We identified TAM on bone marrow sections of MM patients by immunostainings for CD163. A higher number of CD163+ cells was noticed in the MM samples compared to normal samples. Further analysis of the number CD163+cells in healthy areas and MM infiltrated areas confirmed that the higher amount of CD163+ originates from MM infiltrated areas.
In addition, in the 5T33MM mouse model the phenotype of bone marrow located macrophages was investigated. Macrophages isolated from both 5T33MM and naive mice revealed a different expression of several macrophage surface markers: a higher expression of CD206, CD80 and CD115; a lower expression of CD86, CCR7, Ly6G and MHCII on the membrane of 5T33MM macrophages compared to naive macrophages. According to these data, macrophages isolated from 5T33MM mice had more frequently an M2 profile (CD206+) compared to macrophages isolated from naive mice (pNext, we generated TAMs in vitro. CD11b+ cells, isolated from naive mice, were incubated with conditioned medium (CM) from 5T33MMvitro cells. After 1 week culture, the phenotype of surviving cells was analyzed. The bulk population was positive for the macrophage marker F4/80. Also a higher expression of CD206 was observed on generated TAMs compared to the starting population. 5T33MMvitro CM contains Interleukin-10 (IL-10) which is described as a M2 macrophage differentiator. Addition of IL-10 blocking antibodies reduced CD206 upregulation on generated TAMs with an average of 30% compared to control.
The involvement of TAMs in drug resistance was investigated. In vitro generated TAMs were cocultured with 5T33MMvt cells with or without bortezomib or melphalan. TAMs increased the survival of 5T33MMvt cells compared to control (pPreliminary results performed with ex vivo isolated macrophages from 5T33MM mice, also demonstrated a protection from apoptosis of 5T33MMvt cells when co-cultured with ex vivo isolated macrophages. This protective effect remains when bortezomib 5-10nM or Melphalan 15µM were added.
In conclusion, we analyzed the presence and characteristics of macrophages in MM. The number of macrophages is higher in bone marrow samples from MM patients compared to healthy persons. Also, a higher percentage of M2 macrophages (CD11b+/F4/80+/CD206+) was observed in the bone marrow of 5T33MM mice compared to naive mice. In vitro generation of M2 macrophages with 5T33MMvt CM is at least partially IL-10 mediated. Furthermore, MM-associated macrophages contribute to a better survival of 5T33MMvt cells and protect them against several anti-myeloma drugs.