Antineoplastic Drug NSC631570 Inhibits C6 Glioma Growth in Rats Through the Re-education of

1. Antineoplastic Drug NSC631570 Inhibits C6 GliomaGrowth in Rats Through the Re-education of Tumor-Associated Microglia Larysa Skivka, Mariia Rudyk, Ievgenia Opeida, Vitalina Svyatetska, Kateryna Stepura, Niccola Funel, Ascold Nowicky,Wassil Nowicky Taras Sevchenko National University of Kyiv, Ukraine Ukrainian Anticancer Institute, Vienna, Austria University of Pisa, Pisa, Italy

2. Glioma accounts for ~80% of central nervous system tumors. • Glioblastoma multiforme is the most common type of primary brain cancer. • Glioblastoma is resistant to standard therapy. • It is also the most aggressive and lethal tumor. Given the diagnosis, the median survival time is approximately 14 months even with intensive treatment. Less than 5% of patients survive beyond the third year. • Number of newly diagnosed cases of glioblastoma is expected to increase in US, Germany, France and Japan.

3. Hallmark of most solid tumors is an infiltration with immune cells. • Mononuclear phagocytes are most abundant immune cells in tumor microenvironment. Melissa R.Juntilla & Frederic J.de Sauvage, 2013

4. Glioblastoma is heavily infiltrated with myeloid cells (brain-resident microglia and peripheral phagocytes), that are collectively referred to as glioma (glioblastoma) - associated microglia/macrophages or “GAMs”. Brain-resident microglia Peripheral phagocytes Tumor cells Tumor-supressive Pro-tumoral GAMs • NO • pro-inflammatory cytokines • arginase • anti-inflammatory cytokines

5. Tumor-associated hypoxia potentiates GAM alternative polarization. Normoxic proliferating tumor cells Hypoxic area of tumor Hypoxic quiescent tumor cells Tumor-associated macrophages: recruited in tumor growth area and polarized to M2 phenotype by hypoxia and tumor-derived cytokines Tumor vasculature

6. Effects of GAM depletion or re-education on experimental glioblastoma (Mario Leonardo Squadrito & Michele de Palma, 2015)

7. Agents that are used for the re-education of tumor-associated phagocytes in the cancer treatment

8. IDEA: To use NSC631570 for the GAM reprogramming in rats with experimental glioblastoma

9. NSC631570 is an anticancer agent created on the basis of alcaloids from the plant Chelidonium majusL. • It can selectively accumulate in tumor cells and induce their death without damaging healthy cells. • ULA-DC test confirmed that tumor cells consumed much more drug than non-malignant cells in vitro. • (Funel N. et al., 2010)

10. The drug induces immunogenic tumor cell death (Skivka et al., 2014) Damage-associated molecular patterns (alarmins) HMGB1 NSC631570 Tumor cell Cell death Immune cell activation • NSC631570 induces apoptosis in human cultured glioblastoma cells(Gaglianoet al., 2006; Gaglianoet al., 2007)

11. NSC 631570 can influence macrophage migration and cause an influx of macrophages into the site of its injection. • (Korolenko et al., 1998) • NSC 631570 causes an influx of macrophages into the tumor growth area after intravenous administration. • (Skivka et al., 2012) • The preparation stimulates lysosome-phagosome fusion in intact mouse peritoneal macrophages in vitro. (Mozhenoket al., 2005) • NSC 631570 induces M1 functional polarization of peritoneal macrophages in tumor-bearing mice.(Grinevych et al., 2005;Skivka et al., 2011) • NSC 631570 can restore pro–inflammatory functions of macrophages, alternatively polarized by hypoxia. • (Skivka et al., 2013)

12. AIM: to investigate the effect of NSC631570 on glioblastoma-associated phagocyte metabolic profile

13. In vitro experiment design control Normoxia, 21% O2 • Arginase activity • NO production Hypoxia, 3% O2 control 24 h 48 h Arginine metabolism examination Arginine metabolism examination

14. In vivo experiment design C6 glioma cell transplantation using intracranial catheter (day 0) • Examination of GAM functional profile (day 23) : • Flow cytometry • Phagocytosis of S.aureus • ROS generation • CD11b, CD14 and CD206 expression • Arginine metabolism • Arginase activity • NO production NSC631570 introduction 1 4 7 10 13 16 19 days Groups of animals (n=8 per group) Group 1 – control intact rats Group 2 – catheterization Group 3 – C6 glioma Group 4– C6 glioma + NSC631570

15. Tumor-supressive (M1) GAM Pro-tumoral (M2) GAM Arginase activity NO production ROS generation ( –?) Phagocytosis down-regulated up-regulated CD14 expression CD11b expression down-regulated up-regulated CD206 expression up-regulated (?) up-regulated (?)

16. Arginase activity (A) and NO synthesis (B) in rat normoxic and hypoxic microglia cells after the treatment with NSC631570 *– р≤0,05 compared with untreated cells

17. Survival (A) and body weight (B) of rats with C6 glioma after the treatment with NSC631570 *– р≤0,05 compared with control group; # – р≤0,05 compared with C6 glioma group

18. Microglia arginine metabolism in rats with C6 glioma after the treatment with NSC631570 Arginase activity NO production # – р≤0,05 compared with C6 glioma group

19. Microglia reactive oxygen species generation in rats with C6 glioma after the treatment with NSC631570 *– р≤0,05 compared with control group; # – р≤0,05 compared with C6 glioma group

20. Microglia phagocytosis in rats with C6 glioma after the treatment with NSC631570 The number of CD11b+ phagocytizing cells, % Phagocytosis intensity *– р≤0,05 compared with control group; # – р≤0,05 compared with C6 glioma group

21. CD11b and CD14 expression in microglia from rats with C6 glioma after the treatment with NSC631570 *– р≤0,05 compared with control group; # – р≤0,05 compared with C6 glioma group

22. CD206 expression in microglia from rats with C6 glioma after the treatment with NSC631570 “...this (CD206) purported M2 marker did not correlate with microglia immune-suppressive functional indices...” (Gabrusiewicz et al., 2017) ...the use of single phenotypic marker CD206 is not sufficient to identify microglia polarization...CD206 is up-regulated in M2- and M1-skewed microglia... (Wu et al., 2017) *– р≤0,05 compared with control group; # – р≤0,05 compared with C6 glioma group.

23. CONCLUSIONS • NSC631570 repolarize arginine metabolism in hypoxic microglial cells; • treatment of C6 glioma-bearing rats with causes the influx of mononuclear phagocytes into the brain; • intracranial introduction of NSC631570 is accompanied by the sharp increase of NO production by glioma-associated phagocytes; • locoreginal administration of NSC631570 to C6 glioma-bearing animals is associated withup-regulated expression of microglia/macrophage functional maturity (activation) markers: CD11b, CD14, CD206.

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