Trabectedin and Lurbinectedin Extend Survival of Mice Bearing C26 Colon Adenocarcinoma, without Affecting Tumor Growth or Cachexia

Trabectedin (ET743) and lurbinectedin (PM01183) limit the production of inflammatory cytokines that are elevated during cancer cachexia. Mice carrying C26 colon adenocarcinoma display cachexia (i.e., premature death and body wasting with muscle, fat and cardiac tissue depletion), high levels of inflammatory cytokines and subsequent splenomegaly. We tested whether such drugs protected these mice from cachexia. Ten-week-old mice were inoculated with C26 cells and three days later randomized to receive intravenously vehicle or 0.05 mg/kg ET743 or 0.07 mg/kg PM01183, three times a week for three weeks. ET743 or PM01183 extended the lifespan of C26-mice by 30% or 85%, respectively, without affecting tumor growth or food intake. Within 13 days from C26 implant, both drugs did not protect fat, muscle and heart from cachexia. Since PM01183 extended the animal survival more than ET743, we analyzed PM01183 further. In tibialis anterior of C26-mice, but not in atrophying myotubes, PM01183 restrained the NF-κB/PAX7/myogenin axis, possibly reducing the pro-inflammatory milieu, and failed to limit the C/EBPβ/atrogin-1 axis. Inflammation-mediated splenomegaly of C26-mice was inhibited by PM01183 for as long as the treatment lasted, without reducing IL-6, M-CSF or IL-1β in plasma. ET743 and PM01183 extend the survival of C26-bearing mice unchanging tumor growth or cachexia but possibly restrain muscle-related inflammation and C26-induced splenomegaly.

. PM01183 does not prevent fiber atrophy of the gastrocnemii. Representative Hematoxylin and Eosin-stained images of transversal sections of gastrocnemii from mice injected with PBS, C26 vehicle or C26 PM01183 (C26 PM) are shown (scale bar: 50μm) (A). The mean cross-sectional area (CSA) of fibers of 3-4 muscles per group was measured manually with ImageJ software (B). All results are plotted as mean ± SEM, n = 287-994. **** p≤0.0001, Kruskal-Wallis with post hoc Dunn's multiple comparison test (B).

Figure S2. Effect of different doses of PM01183 on viability of fully differentiated myotubes, after 24-hour treatment.
Cell viability was measured with SRB assays and expressed as percentage of controls, represented by corresponding vehicle-treated cells. Results are plotted as mean ± SEM. n = 8. ** p≤0.01 and **** p≤0.001, one-way ANOVA with post-hoc Dunnett's multiple comparison test. Figure S3. PM01183 does not directly prevent C26-induced STAT3 activation in cachectic myotubes. Representative images of myotubes treated with vehicle or 1 nM PM01183 or 33% C26 conditioned medium (S-33%) or in combination (S-33% + PM) for 24 h are shown (scale bar: 100 µ m) (A). Myotube diameters were measured manually with ImageJ software, n = 22-43 (B). Cell viability was measured with SRB assays and expressed as percentage of controls, represented by corresponding vehicle-treated cells, n = 6 (C). Western Blot analysis for pNF-κB/NF-κB, pSTAT3/STAT3 is shown (D) and related band quantitation is plotted, n = 3 (E and F). Vinculin was used as loading control. Representative images of myotubes treated with vehicle or 1 nM PM01183 or with medium conditioned by C26 cells cultured in the Transwell system (TSW) or in combination (TSW + PM) for 24 h are shown (scale bar: 100 µ m) (G). Myotube diameters were measured manually with ImageJ software, n = 41-79 (H). Western Blot analysis for pNF-κB/NF-κB, pSTAT3/STAT3 is shown (I) and related band quantitation is plotted, n = 3 (L and M). Vinculin was used as loading control. Results are plotted as mean ± SEM. * p≤0.05, ** p≤0.01, *** p≤0.001 and **** p≤0.0001, one-way ANOVA with post-hoc Tukey's multiple comparison test.

Hematoxylin and Eosin staining
Ten-μm thick cryosections of gastrocnemius were used for Hematoxylin and Eosin staining. Slides were incubated with Hematoxylin solution for 10 min to stain the nuclei. After a wash with running water, slides were incubated with Eosin solution for 3 min. Finally, slides are washed with 70% ethanol for 20 sec, 90% ethanol for 20 sec, 100% ethanol for 1 min and xylene for 3 min and then, when dried, mounted with a xylene-based mounting medium. Pictures were acquired with a Virtual Slide Microscope VS120 (40X magnification for gastrocnemius, Olympus, Shinjuku, Japan). Similar staining was performed for spleens.

Fiber Size Measurement
Ten-μm thick cryosections of gastrocnemius were used for cross sectional area (CSA) measurements in blind conditions by at least two operators using ImageJ software (National Institutes of Health, Bethesda, MA, USA). Pictures of muscle fibers were acquired with an Olympus Microscope IX71 (20× magnification, 10× ocular lens, Olympus, Shinjuku, Japan) with Cell F (2.6 Build1210, Olympus, Shinjuku, Japan) imaging software for Life Science microscopy (Olympus Soft Imaging solution GmbH, Munster, Germany).

Cell Culture
On the fourth day of differentiation, myotubes were treated for 24 h with different doses of PM01183 (from 0.5 to 20 nM) and toxicity levels were evaluated by sulforhodamine B (SRB) assay (Sigma, St. Louis, MO, USA), using DMSO as vehicle. To further investigate the effect of PM01183 in vitro, myotubes were treated on the fourth day of differentiation for 24h with 1 nM PM01183 or 33% C26 conditioned medium or in combination. C26 cells were seeded at 17000 cells/cm 2 72 h before supernatant collection and grown in differentiation medium for the last 24 h. To characterize the effect of C26 cells on myotubes also in presence of PM01183, we used a Transwell (Corning, NY, USA) co-culture system (as in [1]). C26 cells were seeded at 17000 cells/cm 2 in the Transwell insert 72 h before transfer on fully differentiated myotubes and grown in differentiation medium for the last 48 h. Myotubes with C26 cell insert were treated for 24 h with vehicle or 1 nM PM01183.

Cytokine Measurements
G-CSF and GM-CSF levels in plasma were measured with MILLIPLEX MCYTOMAG-70K-1 from Merck Millipore (Burlington, MA, USA). The detection range is 3.2-10000 pg/mL. Values below the detection limit were not included in the analyses and related graphs. HMGB-1 levels in murine plasma were measured in an enzyme-linked immunosorbent assay (ELISA), according to manufacturer's protocol (EM0382, FineTest, Wuhan, Hubei, China). Values below the detection limit were not included in the analyses and related graphs. The detection range is 15.625-1000 pg/mL.