Bone marrow-derived mesenchymal stromal cells promote survival and drug resistance in tumor cells

Abstract

Bone marrow mesenchymal stromal cells (BMMSC) have antitumorigenic activities. Here, we hypothesized that circulating BMMSC are incorporated into tumors and protect tumor cells from therapy-induced apoptosis. Adherent cells harvested from murine bone marrow and expressing phenotypic and functional characteristics of BMMSC were tested for their antitumor activity against murine 4T1 mammary adenocarcinoma and LL/2 Lewis lung carcinoma cells. BMMSC but not NIH3T3 or murine skin fibroblasts stimulated the expansion of 4T1 cells in three-dimensional (3D) cocultures, and conditioned medium (CM) from these cells increased the viability of 4T1 and LL/2 cells in two-dimensional (2D) cultures. 4T1 cells exposed to BMMSC CM exhibited a 2-fold reduction in apoptosis under low serum concentrations (0.5% to 1%). Furthermore, exposure of 4T1 and LL/2 cells to BMMSC CM increased their viability in the presence of paclitaxel or doxorubicin at therapeutic concentrations. This effect was accompanied by reductions in caspase-3 activity and Annexin V expression. When coinjected with 4T1 cells in the mammary fat pad of mice subsequently treated with doxorubicin, BMMSC (and not fibroblasts) also inhibited drug-induced apoptosis in tumor cells by 44%. We demonstrated that BMMSC were attracted by 4T1 and LL/2 cells but not by NIH3T3 cells in vitro and that when injected intravenously in 4T1 tumor-bearing mice, these cells (and not NIH3T3) were specifically detected in tumors within 12 to 18 days in which they preferentially localized at the invasive front. Overall, our data identify BMMSC as an important mediator of tumor cell survival and treatment resistance in primary tumors.

Figure 1

Primary murine bone marrow stromal isolates display mesenchymal cell markers. A, FACS analysis of BMMSC surface markers. Representative histograms for early passages (<10) shown (open histogram represents the autofluorescent control). B, spontaneous osteoblast/adipocyte differentiation of BMMSC in culture. Upper panel: light micrographs of early passage BMMSC cultured for 2–3 weeks and stained for alkaline phosphatase (AP) activity (osteoblasts) or lipids by Oil Red O (ORO, adipocytes). Scale bar = 250 μm (left) and 100 μm (right). Lower panel: percent of BMMSC with positive staining for AP and ORO over passages in culture. The data represent the mean ±SD of positive cells counted in five 40x fields. C, mature osteoblast differentiation of BMMSC cultured 3 weeks in osteogenesis medium vs. control medium and stained with Alizarin red. D, early adipocyte differentiation of BMMSC cultured for 2 weeks in maintenance medium or switched to adipogenic medium after 1 week, and stained for the presence of lipids with ORO (Scale bar = 100 μm).

Figure 2

BMMSC enhance tumor cell growth by suppressing spontaneous apoptosis. A, Matrigel-embedded 4T1 cells cultured for 21 days in the presence or absence of BMMSC, NIH3T3 cells, or normal mouse skin fibroblasts in a transwell chamber were examined for the formation of tumorspheres. The data represent the mean ±SD number of 4T1 tumorspheres in quadruplicate wells from one of two experiments showing similar results. Inset: optical light micrographs of 4T1 spheroids in the central field of representative control and co-culture wells. Scale bar = 500 μm. B, 4T1 cells were cultured in 2D for 4 days in the presence or absence of transwells containing BMMSC, NIH3T3, or fibroblasts (left panel) or in the presence or absence of CM from BMMSC, NIH3T3, or fibroblasts (right panel). The data represent the mean ±SD of viable cells at indicated times from triplicate wells. C, 4T1 cells were cultured in 2D for 5 days in the presence or absence of CM from BMMSC or NIH3T3 cells. Top panel: representative analysis of apoptosis by flow cytometry at day 5. Lower panel: the data represent the mean ±SD of Annexin V-FITC positive cells at indicated times in triplicate wells from one of two experiments showing similar results. D, 4T1 cells were cultured for 5 days in the presence or absence of CM from BMMSC and examined for caspase activity at the indicated times. The data represent the mean ±SD of caspase-3 (top), caspase-9 (middle), and caspase-8 (bottom) activity at day 3, 4, and 5 from triplicate wells from one of two experiments showing similar results.

Figure 3

BMMSC enhance survival in drug-treated tumor cells. A, cell viability of 4T1 cells (1×10⁴) cultured in the presence or absence of 0.5% serum BMMSC-derived CM and treated with paclitaxel (upper panel) or doxorubicin (lower panel) for 96 hours. The data represent the mean ±SD percentage of surviving cells for indicated drug concentrations in triplicate wells from one of three experiments showing similar results. B, cell viability of LL/2 cells (1×10⁴) cultured and treated as indicated in A for 72 hours. The data represent the mean ±SD percentage of surviving cells for indicated drug concentrations in triplicate wells from one of two experiments showing similar results.

Figure 4

BMMSC CM protects tumor cells from drug-induced apoptosis. A, 4T1 cells (1×10⁴) cultured in the presence or absence of 0.5% serum BMMSC CM and treated with paclitaxel (upper panel) or doxorubicin (lower panel) at indicated concentrations for 24 hours were examined for caspase-3/7 activity. The data represent the mean ±SD fold-change from 4T1 cells cultured in the presence of regular medium (controls) in triplicate wells. They are representative of two experiments showing similar results. B, LL/2 cells (1×10⁴) cultured as indicated in A were examined for caspase 3/7 activity. The data represent the mean ±SD fold-change from controls in triplicate wells from one of two experiments showing similar results. C, 4T1 cells cultured as indicated in A were examined for Annexin V expression by flow cytometry after 16 and 24 hours. Left panel: the data represent the mean ±SD of Annexin V-FITC positive cells in triplicate wells. Right panel: representative analysis by flow cytometry at 16 hours.

Figure 5

BMMSC suppress apoptosis in drug-treated tumors in vivo. 4T1 cells (1×10⁶) were injected in the mammary fat pad of Balb/cJ mice in the absence or presence of 2×10⁵ BMMSC or normal mouse fibroblasts. Three days post-injection of tumor cells, mice were treated with doxorubicin for two consecutive days, and tumors were harvested on day 5 and examined for the presence of apoptotic E-cadherin-positive cells. A, fluorescent micrographs of representative tumor sections from control and tumors co-injected with BMMSC or fibroblasts, stained for the presence of E-cadherin and apoptotic nuclei. Top: TUNEL, middle: E-cadherin, and bottom: overlay with DAPI. Arrowheads indicate the presence of apoptotic nuclei. Scale bar = 50 μm. B, the data represent the mean ±SD of TUNEL-positive, E-cadherin-positive cells counted in a total of 5 32x fields in each of 5 tumors.

Figure 6

BMMSC exhibit a chemotactic response to tumor cells in vitro and in vivo. A, BMMSC (8×10⁴ per well) were cultured for 3 hours or 48 hours in a transwell culture well in the presence or absence of serum-free CM from 4T1, LL/2, and NIH3T3 cells. Left panel: the data represent the mean ±SD number of migrated BMMSC from triplicate filters from one of two separate experiments showing similar results. Right panel: light micrographs of the bottom side of representative transwell filters from 48 hour assays. Scale bar = 100 μm. B, fluorescence analysis of frozen sections (6 μm) of 4T1 tumors and indicated organs harvested on day 14 post injection of tumor cells (6×10⁵ injected s.c. in the left flank) and day 2 post-injection of PKH26-labeled BMMSC (2×10⁶ via tail vein). Cy3/DAPI overlay for tumor, liver, lung, and kidney are shown. Scale bar = 50 μm. C, histological analysis of iron deposits by Prussian blue staining on sections from FFPE 4T1 tumors (6 μm) harvested on day 10 of tumor growth (5×10⁵ injected s.c. in the left flank) and 6 days post-injection of iron-labeled BMMSC (3×10⁵ via tail vein). Scale bar = 250 μm (left) and 30 μm (right). T = tumor; N = normal tissue.

Figure 7

BMMSC are specifically localized to tumors. Luciferase-expressing BMMSC or NIH3T3 cells (2×10⁶) were injected i.v. into Balb/cJ mice bearing 4T1 tumors (n= 5; 2×10⁶ cells injected s.c. in the left flank) on day 2 of tumor growth. A, serial bioluminescent imaging of BMMSC and NIH3T3-injected mice over time (at indicated days post BMMSC/NIH3T3 injection). B, quantification of luminescent signals from tumor and normal organ lysates obtained from mice sacrificed on day 18 post BMMSC/NIH3T3 injection. The data represent the luminescent signal (RLU) of 10 mg of tissue lysate, with SD for 2 replicates shown.