Lurbinectedin induces depletion of tumor-associated macrophages, an essential component of its in vivo synergism with gemcitabine, in pancreatic adenocarcinoma mouse models

Abstract

We explored whether the combination of lurbinectedin (PM01183) with the antimetabolite gemcitabine could result in a synergistic antitumor effect in pancreatic ductal adenocarcinoma (PDA) mouse models. We also studied the contribution of lurbinectedin to this synergism. This drug presents a dual pharmacological effect that contributes to its in vivo antitumor activity: (i) specific binding to DNA minor grooves, inhibiting active transcription and DNA repair; and (ii) specific depletion of tumor-associated macrophages (TAMs). We evaluated the in vivo antitumor activity of lurbinectedin and gemcitabine as single agents and in combination in SW-1990 and MIA PaCa-2 cell-line xenografts and in patient-derived PDA models (AVATAR). Lurbinectedin-gemcitabine combination induced a synergistic effect on both MIA PaCa-2 [combination index (CI)=0.66] and SW-1990 (CI=0.80) tumor xenografts. It also induced complete tumor remissions in four out of six patient-derived PDA xenografts. This synergism was associated with enhanced DNA damage (anti-γ-H2AX), cell cycle blockage, caspase-3 activation and apoptosis. In addition to the enhanced DNA damage, which is a consequence of the interaction of the two drugs with the DNA, lurbinectedin induced TAM depletion leading to cytidine deaminase (CDA) downregulation in PDA tumors. This effect could, in turn, induce an increase of gemcitabine-mediated DNA damage that was especially relevant in high-density TAM tumors. These results show that lurbinectedin can be used to develop 'molecularly targeted' combination strategies.

Keywords: Gemcitabine; Lurbinectedin; PDA mouse models; Synergism; Tumor-associated macrophage depletion.

Conflict of interest statement

P.Av., C.C., C.M.G. and M.J.G. are employees and shareholders of PharmaMar, SA (Madrid, Spain). No potential conflicts of interest were disclosed by the other authors.

© 2016. Published by The Company of Biologists Ltd.

Figures

Fig. 1.

In vivo characterization of the synergistic effect of lurbinectedin (PM01183) combined with gemcitabine. Nude athymic mice bearing subcutaneous tumors (SW-1990 or MIA PaCa-2) sized ca. 150 mm3 were randomly allocated and treated with PM01183 (0.180 mg kg−1), gemcitabine (180.0 mg kg−1) or its combination [PM01183 plus gemcitabine (0.180 mg kg−1+180.0 mg kg−1)]. (A,B) Tumor growth (median) curves for mice bearing SW-1190 (A) or MIA PaCa-2 (B) tumors and treated with the highest doses of PM01183, gemcitabine or its combination. (C,D) Antitumor activity of each single or combined high-dosed treatment followed by T/C values, defined as the change in tumor volume for each treated (T) and placebo (C) group during the placebo-treated survival period for mice bearing SW-1190 (C) or MIA PaCa-2 (D). (E,F) Determination of tumor fraction affected (Fa) by treatments, calculated according to the formula Fa=1– T/C and combination index (CI) determined by the CI-isobol method for mice bearing SW-1190 (E) or MIA PaCa-2 (F). Statistically significant differences at P<0.01 (two-tailed Mann–Whitney U-test).

Fig. 2.

DNA damage in tumor-bearing mice after treatment with the lurbinectedin (PM01183)-gemcitabine combination. (A) Representative γ-H2AX-stained nuclei of SW-1990 (left panels) or MIA PaCa-2 (right panels) tumors 24 h after the administration of placebo, PM01183 (0.180 mg kg−1), gemcitabine (180.0 mg kg−1) or the combination (PM01183 plus gemcitabine, 0.180 mg kg−1+ 180.0 mg kg−1). (Original magnification, ×400.) (B,C) Quantitation of the number of γ-H2AX nuclei per µm2 in SW-1990 (B) or MIA PaCa-2 (C) tumors. Statistically significant differences at *P<0.01 (two-tailed Mann–Whitney U-test).

Fig. 3.

Caspase-3 activation and apoptosis induction in tumor-bearing mice after treatment with the lurbinectedin (PM01183)-gemcitabine combination. Representative immunohistochemistry (IHC) micrographs of cleaved (active) caspase-3 and apoptotic induction by Hoechst staining in SW-1990 (A) or MIA PaCa-2 (B) tumors, 24 h after the administration of placebo, PM01183 (0.180 mg kg−1), gemcitabine (180.0 mg kg−1) or the combination (PM01183 plus gemcitabine, 0.180 mg kg−1+180.0 mg kg−1). (Original magnification, ×400.) Arrows indicate apoptotic cells. (C,D) Quantitation of the number of cleaved-caspase-3-positive cells per µm2 in SW-1990 (C) or MIA PaCa-2 (D) tumors. Statistically significant differences at *P<0.01 (two-tailed Mann–Whitney U-test).

Fig. 4.

Lurbinectedin (PM01183) induction of proliferative block or macrophage depletion and cytidine deaminase (CDA) downregulation, as a single agent or combined with gemcitabine. Representative micrographs of proliferation rate (anti-Ki67 antibody staining), tumor associated macrophages (TAM; staining with an anti-F4/80 antibody) and CDA expression in tumor xenografts of SW-1990 (A) or MIA PaCa-2 (B) 24 h after the administration of placebo, PM01183 (0.180 mg kg−1), gemcitabine (180.0 mg kg−1) or the combination (PM01183 plus gemcitabine, 0.180 mg kg−1+180.0 mg kg−1). Gemcitabine treatment upregulates CDA. PM01183 induces TAM depletion and CDA upregulation in MIA PaCa-2 tumors, whereas it induces a proliferative block in SW-1990 tumors (original magnification 400×).

Fig. 5.

Differences in macrophage staining and cytidine deaminase (CDA) expression among treatment groups. Graphs depicting the number of tumor associated macrophages (TAMs; A,C) and cells with CDA expression (B,D) per µm2 in the complete tumor area of SW-1990 (A,B) or MIA PaCa-2 (C,D) mice 24 h after the administration of placebo, lurbinectedin (PM01183, 0.180 mg kg−1), gemcitabine (180.0 mg kg−1) or the combination (PM01183 plus gemcitabine, 0.180 mg kg−1+180.0 mg kg−1) as assessed by immunohistochemical staining. See representative images in Fig. 4. Statistically significant differences at *P<0.01 (two-tailed Mann–Whitney U-test).

Fig. 6.

Tumor response to treatment in athymic nude mice bearing PDA-AVATAR xenografts. Mice bearing Panc-026, Panc-265, Panc-354, Panc-291, JH-010 or JH-024 tumors were treated with placebo, lurbinectedin (PM01183, 0.180 mg kg−1) or gemcitabine (180.0 mg kg−1) on days 0, 7, 14, 21 and 28. Treatment-induced antitumor activity was determined by the relative ΔT/ΔC (%), defined as the percentage of change in tumor size for treated (T) and placebo (C) groups in each experiment. Also, complete tumor regressions (CR) were defined when two or more consecutive tumor measurements were smaller than 63 mm3. DPC4, MADH4/SMAD4 gene.