Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach - PubMed (original) (raw)

Comparative Study

. 2006 Aug 7;95(3):272-81.

doi: 10.1038/sj.bjc.6603240. Epub 2006 Jul 11.

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Comparative Study

Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach

S M Zeisberger et al. Br J Cancer. 2006.

Abstract

Tumour-associated macrophages, TAMs, play a pivotal role in tumour growth and metastasis by promoting tumour angiogenesis. Treatment with clodronate encapsulated in liposomes (clodrolip) efficiently depleted these phagocytic cells in the murine F9 teratocarcinoma and human A673 rhabdomyosarcoma mouse tumour models resulting in significant inhibition of tumour growth ranging from 75 to >92%, depending on therapy and schedule. Tumour inhibition was accompanied by a drastic reduction in blood vessel density in the tumour tissue. Vascular endothelial growth factor (VEGF) is one of the major inducers of tumour angiogenesis and is also required for macrophage recruitment. The strongest effects were observed with the combination therapy of clodrolip and a VEGF-neutralising antibody, whereas free clodronate was not significantly active. Immunohistologic evaluation of the tumours showed significant depletion of F4/80+ and MOMA-1+ and a less pronounced depletion of CD11b+ TAMs. Blood vessel staining (CD31) and quantification of the vessels as well as TAMs and tumour-associated dendritic cells (TADCs) in the A673 model showed reduction rates of 85 to >94%, even 9 days after the end of therapy. In addition, CD11c+ TADCs, which have been shown to potentially differentiate into endothelial-like cells upon stimulation by tumour released growth and differentiation factors, were similarly reduced by clodrolip or antibody treatment. These results validate clodrolip therapy in combination with angiogenesis inhibitors as a promising novel strategy for an indirect cancer therapy aimed at the haematopoietic precursor cells that stimulate tumour growth and dissemination and as a tool to study the role of macrophages and dendritic cells in tumorigenesis.

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Figures

Figure 1

Figure 1

In vitro and in vivo effects of free and liposome encapsulated clodronate (clodrolip). (A) Concentration-dependent cytotoxicity of clodrolip on macrophages (isolated from Sv129 mice by peritoneal lavage) in vitro. (B) Cytotoxicity of clodronate or clodrolip on different cells in vitro. Macrophages, HUVE, F9 and A673 cells were cultured in the presence of 1 mg ml−1 clodronate or clodrolip for 6 h. Results are means±s.e.m. (_n_=3). Statistical analysis: *P<0.05 vs untreated cells. (C) Selective depletion of spleen cell populations after treatment with clodronate and clodrolip. Spleen tissues obtained from immunocompetent Sv129 mice injected with PB, clodronate or with clodrolip are shown (initial dose 2 mg 20 g−1 mouse body weight, followed by 1 mg, every 4 days, i.p.). Spleens were removed and sections IHC stained for marginal zone metallophilic MOMA1+, marginal zone ER-TR 9+, red pulp F4/80+, CD68+ and CD11b+ macrophages, the DC subsets FDC+ and CD11c+, B220+ B cells, and CD3+ T cells. Bar: 100 _μ_m.

Figure 2

Figure 2

Effects of clodronate treatment on F9 teratocarcinoma growth. (A) Tumour-bearing mice (6–8 Sv129 mice group−1) were treated by i.p. injection starting 6 h after tumour cell inoculation, followed by treatments every 4 days (days 0, 4, 8 and 12) at the same dosage as in Figure 1B). In two groups, clodrolip therapy was started on days 4 or 8 after tumour cell inoculation. Values represent tumour growth±s.e.m. (_n_=6–8). Relative percentual tumour growth was normalised to day 1. (B) Comparison of clodronate with clodrolip treatment in the A673 rhabdomyosarcoma model. Tumour-bearing mice (6–8 CD-1 nude mice group−1) were treated four times every 4 days starting on day 1 (days 1, 5, 9 and 13, i.p.). Treatment doses were as in Figure 1B for the high-dose (HD) groups. The dosage for low-dose (LD) groups was 1 mg 20 g−1 mouse body weight as initial dose, followed by 0.5 mg for the subsequent doses. Values shown represent tumour growth±s.e.m. (_n_=6–8). Relative percentual tumour growth was normalised to day 1. (C) Immunohistochemical quantification of F4/80+, MOMA1+ TAMs and CD31+ endothelial cells (blood vessels) in A673 tumours. Three to five tumour sections were stained with the corresponding antibodies and F4/80+, MOMA1+ and CD31+ areas in three randomly selected fields of each section were quantified. Bars indicate the calculated averages±s.e.m. (_n_=9–15) in percent referred to the PB control sections. Statistical analysis: *P<0.05; **P<0.01 and ***P<0.001. Individual _P_-values: F4/80, clodrolip HD, **_P_=0.0032; clodrolip LD, *_P_=0.0138; clodronate HD and LD, NS and combination LD+LD, **_P_=0.01. MOMA1, clodrolip HD and LD, ***P<0.0001; clodronate HD, ***_P_=0.0005; clodronate LD, **_P_=0.0014 and combination LD+LD, ***P<0.0001. CD31, clodrolip HD and LD, ***P< 0.0002, clodronate HD and LD and combination LD+LD, NS and clodrolip HD or LD vs clodronate HD, ***P<0.0003.

Figure 3

Figure 3

Combination of clodrolip treatment with VEGF neutralisation further enhances TAM depletion and shows improved tumour inhibition in the syngeneic F9 teratocarcinoma (A, B) and in the xenogenic human A673 rhabdomyosarcoma (C, D) models. (A) Tumour growth inhibition by clodrolip treatment in combination with the anti-mVEGF Ab V65. F9 tumour-bearing mice (3–5 group−1) were treated with PB, control Ab A1, V65, clodrolip plus A1 or clodrolip plus V65. Each clodrolip dose contained initially 2 mg 20 g−1 mouse body weight (day 0, i.p.), followed by 1 mg (days 6 and 11, i.p) and the Abs were given at 0.5 mg (days 3–7 and 10, i.v.). Values represent the mean of treated mice±s.e.m. (_n_=3–5). Relative percentual tumour growth was normalised to day 1. (B) Bar graph of tumour volumes measured on day 14, showing the calculated averages±s.e.m. (_n_=3–5). Statistical analysis: *P<0.05; V65 vs clodrolip+V65, _P_=0.0118 and clodrolip+A1 vs clodrolip+V65, _P_=0.014. (C) Tumour growth inhibition by the combination treatment in A673 rhabdomyosarcomas. Tumour-bearing mice (6–8/group) were treated with PB, control Ab A1, SZH9 Ab (days 5–13, i.v.), clodrolip plus A1 or clodrolip plus SZH9 (days 1, 5, 9 and 13, i.p.) as in (A). Values represent the mean of treated mice±s.e.m. (_n_=6–8). (D) Bar graph of tumour growth inhibition measured at days 16 (open bars) and 22 (grey bars) showing the tumour volumes as calculated averages±s.e.m. Statistical analysis: individual _P_-values for day 16, *_P_=0.039, **_P_=0.015, ***_P_=0.009; for day 22, *_P_=0.026, **_P_=0.007, ***_P_=0.0002; NS, not significant. VEGF-neutralising properties and pharmacokinetic data of SZH9 are shown in Supplementary Figures s4 and s5.

Figure 4

Figure 4

Immunohistochemical analysis of TAM depletion and correlation to blood vessel density. (A) A673 tumour sections obtained from mice injected with PB, A1, SZH9, clodrolip alone or in combination with SZH9 on days 16 after onset of treatment were analysed by IHC. Depletion of activated F4/80+, marginal zone MOMA1+ and CD11b+ TAMs and of CD11c+ tumour-associated dendritic cells (TADCs) is shown. Blood vessel staining is shown by staining of CD31+ endothelial cells. Arrows show cells that were not depleted (or repopulated) after the end of therapy. Bars: 100 _μ_m. (B) Quantification of F4/80+, MOMA1+, CD11b+ TAMs, CD11c+ TADCs and CD31+ ECs (blood vessels) at day 22 (see Figure 3C and D). Three to five tumour sections were stained and quantified as indicated in Figure 2C. The bars show the calculated averages±s.e.m. (_n_=9–15) in percents referred to the PB control sections. Statistical analysis: *P<0.05; **P<0.01 and ***P<0.001. Individual _P_-values: F4/80, SZH9, NS; clodrolip, **_P_=0.0003; clodrolip+SZH9, ***P<0.0001. MOMA1, SZH9, *_P_=0.043; clodrolip, ***_P_=0.0001; clodrolip+SZH9, ***P<0.0001. CD31, SZH9, *_P_=0.03; clodrolip, ***P<0.0001; clodrolip+SZH9, ***P<0.0001. CD11b, SZH9, NS; clodrolip, _P_=0.033; clodrolip+SZH9, *_P_=0.03. CD11c, SZH9, *_P_=0.019; clodrolip, **_P_=0.0055 and clodrolip+SZH9, **_P_=0.0002. (C) Correlation of TAM depletion (F4/80+, MOMA-1+, CD11b+) and of CD11c+ TADCs vs vessel density (CD31+ cells). The dots represent values of positively stained areas from individual tumours, showing the clear separation of clodrolip treated (box) compared to other groups (PB, Ab A1, Ab SZH9). Statistical analysis (Pearson correlation, _n_=20): F4/80, _r_=0.701, _P_=0.0006; MOMA1, _r_=0.711, _P_=0.0004; CD11b, _r_=0.50, _P_=0.025; CD11c, _r_=0.531, _P_=0.016.

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