(A) The ALDEFLUOR-positive population of SUM159 is increased by co-culture with MCs, transwell-culture with MCs, or culture with conditioned medium (C.M.) derived from either co-culture or transwell-culture, but not by culture with C.M. derived from MCs alone. The total cell number of SUM159 cells remains constant under all of the treatment conditions.
(B) The ALDEFLUOR-positive population of MCs is approximately 5% and is not changed by co-culture with either ALDEFLUOR-positive or ALDEFLUOR-negative SUM159 cells.
(C) The ALDEFLUOR-positive but not the ALDEFLUOR-negative population of MCs is capable of osteogenic and adipogenic differentiation.
(D) ALDEFLUOR-positive MCs increase the proportion of ALDEFLUOR-positive SUM159 cells. The p values refer to the significant difference of sample groups compared to the “alone” group. Error bar represent the SD.

Protein level of CXCL5 (ENA78), CXCL6 (GCP2), CXCL1 (Gro-α), CXCL7, IL6 and IL8 is increased by the co-culture of breast tumor cells and MCs. The * p<0.05 refer to the significant difference of the individual cytokine from the “co-culture” group compared to the corresponding cytokine from “A+B” group. Error bar represent the SD.

(A) Recombinant cytokines increase the ALDEFLUOR-positive population of SUM159 cells in the order of CXCL7 > IL6 > IL8 > CXCL6 > CXCL5.
(B) Blocking antibodies decrease the ALDEFLUOR-positive population of SUM159 cells in the co-culture in the order of anti-CXCL7 > anti- IL6 > anti-IL8 > anti-CXCL6 > anti-CXCL5. N.T. represents No Treatments. The p values refer to the significant difference of sample groups compared to the “N.T.” group.
(C) Addition of CXCL7 to SUM159 cells induces a cytokine expression pattern which recapitulated that of co-culture. Alternatively, the addition of CXCL7 blocking antibody to the co-culture completely blocked expression of induced cytokines. The * p<0.05 refer to the significant difference of the individual cytokine from the “co-culture treated with 20ug/ml anti-CXCL7” group compared to the corresponding cytokine from “co-culture” group, or the significant difference of the individual cytokine from the “SUM159 treated with 10ng/ml CXCL7” group compared to the corresponding cytokine from “SUM159 alone” group.
Error bar represent the SD.

(A) Effects of optimized concentration of cytokine on the induction of CXCL7 by MCs. IL6 induce more than a ten-fold increase of CXCL7 production by MCs. N.T. represents No Treatments. The p value refers to the significant difference of the sample group compared to the “N.T.” group.
(B) Both the IL6R and GP130 were primarily expressed in the ALDEFLUOR-positive populations of MCs. The p values refer to the significant difference of the “ALDH⁺” group compared to the “ALDH⁻” group.
(C) MCs were treated with vehicle control or 100 ng/ml of IL6 for 4 days, and both ALDEFLUOR-positive and ALDEFLUOR-negative cells were sorted out from both groups, and total RNA was isolated. Real-time RT-PCR showed that CXCL7 mRNA level was undetectable in the MC control group, but elevated in ALDEFLUOR-positive MCs with IL6 treatment. The p value refers to the significant difference of the “ALDH⁺” cells compared to the “ALDH⁻” cells in the same group.
(D) ALDEFLUOR-positive MCs, but not Aldefluor-negative MCs, have chemotactic capability which is increased by IL6. Chemotaxis of Aldefluor-positive MCs toward to tumor cells is inhibited by IL6 blocking antibody. The p values refer to the significant difference of sample groups compared to the “CONTROL” group.
(E) A model of cytokine networks mediating the interaction between MSCs, breast cancer cells and breast CSCs.
Error bar represent the SD.

(A) ALDEFLUOR-positive (ALDH+) but not ALDEFLUOR-negative (ALDH-) MCs stimulate tumor growth of SUM159 cells in NOD/SCID mice. The * p<0.05 refer to the significant difference of the sample groups compared to the “SUM159-DsRed ALDH⁺ alone (1k)” group.
(B) ALDEFLUOR-positive (ALDH+) but not ALDEFLUOR-negative (ALDH−) MCs increase the ALDEFLUOR-positive population of SUM159 cells in NOD/SCID mice. The p values refer to the significant difference of sample groups compared to the “SUM159 alone” group.
(C) Four-color immunofluorescence staining reveals close apposition between ALDH1-positive MCs and ALDH1-positive SUM159 cells. In order to quantitate this association, we counted 9 fields to determine the frequency of association of ALDH-positive CSCs and MSCs. There are 14 CSC/MSC direct-contacts out of total 38 MSCs and 35 CSCs. This statistically significant positive association demonstrates the non-random distribution of these two cell types in tumor xenografts. SUM159-DsRed: DsRed-labeled SUM159; MC-GFP: GFP-labeled MC; Blue: DAPI staining; Green: DsRed staining; Purple: GFP staining; Red: ALDH1 staining. White arrow: ALDH1+ SUM159; White triangle: ALDH1+ MC.
(D) Serial dilutions of tumor cells isolated from primary tumors of SUM159 alone or SUM159 mixed with MCs were engrafted into secondary NOD/SCID mice. Tumor latency was reduced and tumor growth was increased in the tumors generated from primary tumors of SUM159 mixed with MCs. The * p<0.05 refer to the significant difference of the “SUM159-DsRed from Mix” compared to the “SUM159-DsRed from Alone” group injected with the same number of cells.
Error bar represent the SD.

(A) MCs labeled with luciferase and DsRed were introduced via intra tibia injection. The tumor growth was facilitated by introducing MCs into mouse tibia. The * p<0.05 refer to the significant difference of the “With MC intra-tibia injection” group compared to the “Without MC intra-tibia injection” group.
(B) In situ localization of ALDH1+ MCs and ALDH1+ SUM159 cells identifies a “cancer stem cell niche”. Light imaging reveals presence of DsRed labeled MC in mice with intratibial MC inoculation but not in controls. Brown: DsRed. Immunoflourescence detects DsRed labeled MC (green), ALDH1-positive MC (yellow) and ALDH1-positive SUM159 cells (red) in merged image.
(C) Immunolocalization of ALDH1-positive tumor cells, MCs and endothelial cells in breast cancer clinical specimens. We counted 6 fields to determine the frequency of association of ALDH-positive CSCs and MSCs. There are 21 CSC/MSC direct-contacts out of total 115 MSCs and 33 CSCs. Stain ALDH1 (a stem/progenitor marker), CD105 (a MSC marker and endothelial cell marker), CD31 (an endothelial cell marker), Pan-cytokeratin AE1/AE3 (Pan-CK, an epithelial cell marker) in consecutive serial sections from primary breast tumor. Shown is one representative sample from three independent samples. Red arrow: CSCs (ALDH1⁺CD105⁻CD31⁻Pan-CK⁺); Yellow arrows: MSCs (ALDH1⁺CD105⁺CD31⁻Pan-CK⁻); Green arrows: Endothelial progenitors (ALDH1⁺CD105⁺CD31⁺). Error bar represent the SD.