BackgroundImmune checkpoint inhibitors (ICIs) have significantly changed the oncology clinic in recent years, improving survival expectations in cancer patients. ICI therapy have a broad spectrum of side effects from endocrinopathies to cardiovascular diseases. In this study, pro-inflammatory and pro-fibrotic effects of short-term ICIs therapy in preclinical models were analyzed.MethodsFirstly, in a human in vitro model, human cardiomyocytes co-cultured with hPBMC were exposed to ICIs (with CTLA-4 or PD-1 blocking agents, at 200 nM) for 72 h. After treatment, production of DAMPs and 12 cytokines were analyzed in the supernatant through colorimetric and enzymatic assays. C57/Bl6 mice were treated with CTLA-4 or PD-1 blocking agents (15 mg/kg) for 10 days. Before (T0), after three days (T3) and after treatments (T10), ejection fraction, fractional shortening, radial and longitudinal strain were calculated by using bidimensional echocardiography (Vevo 2100, Fujfilm). Fibrosis, necrosis, hypertrophy and vascular NF-kB expression were analyzed through Immunohistochemistry. Myocardial expression of DAMPs (S100- Calgranulin, Fibronectin and Galectine-3), MyD88, NLRP3 and twelve cytokines have been analyzed. Systemic levels of SDF-1, IL-1β, and IL-6 were analyzed before, during and after ICIs therapy.ResultsRadial and longitudinal strain were decreased after 10 days of ICIs therapy. Histological analysis of NF-kB expression shows that short-term anti-CTLA-4 or anti-PD-1 treatment increased vascular and myocardial inflammation. No myocardial hypertrophy was seen with the exception of the pembrolizumab group. Myocardial fibrosis and expression of galectin-3, pro-collagen 1-α and MMP-9 were increased after treatment with all ICIs. Both anti-CTLA-4 or anti-PD-1 treatments increased the expression of DAMPs, NLRP3 inflammasome and MyD88 and induced both in vitro and in vivo the secretion of IL-1β, TNF-α and IL-6. Systemic levels of SDF-1, IL-1β and IL-6 were increased during and after treatment with ICIs.ConclusionsShort therapy with PD-1 and CTLA-4 blocking agents increases vascular expression of NF-kB, systemic SDF-1, IL-1β, IL-6 levels and myocardial NLRP3, MyD88 and DAMPs expression in preclinical models. A pro-inflammatory cytokine storm was induced in myocardial tissues and in cultured cardiac cells after ICIs therapy. The overall picture of the study suggests new putative biomarkers of ICIs-mediated systemic and myocardial damages potentially useful in clinical cardioncology.

Renal cell carcinoma (RCC) represents the main renal tumors and are highly metastatic. Sunitinib, a recently-approved, multi-targeted Tyrosine Kinases Inhibitor (TKi), prolongs survival in patients with metastatic renal cell carcinoma and gastrointestinal stromal tumors, however a dose related cardiotoxicity was well described. Polydatin (3,4’,5-trihydroxystilbene-3-β-d-glucoside) is a monocrystalline compound isolated from Polygonum cuspidatum with consolidated anti-oxidant and anti-inflammatory properties, however no studies investigated on its putative cardioprotective and chemosensitizing properties during incubation with sunitinib. We investigated on the effects of polydatin on the oxidative stress, NLRP3 inflammasome and Myd88 expression, highlighting on the production of cytokines and chemokines (IL-1β, IL-6, IL-8, CXCL-12 and TGF-β) during treatment with sunitinib. Exposure of cardiomyocytes and cardiomyoblasts (AC-16 and H9C2 cell lines) and human renal adenocarcinoma cells (769‐P and A498) to polydatin combined to plasma-relevant concentrations of sunitinib reduces significantly iROS, MDA and LTB4 compared to only sunitinib-treated cells (P<0.001). In renal cancer cells and cardiomyocytes polydatin reduces expression of pro-inflammatory cytokines and chemokines involved in myocardial damages and chemoresistance and down-regulates the signaling pathway of NLRP3 inflammasome, MyD88 and NF-κB. Data of the present study, although in vitro, indicate that polydatin, besides reducing oxidative stress, reduces key chemokines involved in cancer cell survival, chemoresistance and cardiac damages of sunitinib through downregulation of NLRP3-MyD88 pathway, applying as a potential nutraceutical agent in preclinical studies of preventive cardio-oncology.

Background Atherosclerosis is now recognized as a chronic inflammatory disease. Oxidized low-density lipoprotein (Ox-LDL) is oxidatively modified form of LDL with a key role in induction and progressio of atherosclerosis. Recent findings reported that cardiovascular events (myocarditis and atherosclerosis) were higher after initiation of immune check-point inhibitors (ICIs), potentially mediated by accelerated progression of atherosclerosis. Purpose We evaluated whether ox-LDL-induced apoptosis through toll-like receptor-4 (TLR4)/Nuclear factor κB (NF-κB) signaling pathway and NLRP3 inflammasome during exposure of human cardiomyocytes to nivolumab turning the light on the mechanisms of cell inflammation induced by OxLDL in cardiotoxicity of ICIs. Methods Human fetal cardiomyocytes (HFC cell line) in co-culture with hPBMC, were exposed to clinically relevant concentration of nivolumab (100 nM) alone or combined to OxLDL at 1, 10 and 50 μg/mL for 24h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis and apoptosis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome, expression of TLR4 and NF-kB). In order to evaluate the pathways involved in OxLDL damages, TLR4 and NLRP3 inhibitor (TAK-242 and dapansutrile, respectively) were added during cell viability and apoptosis studies. Results Nivolumab exerts cytotoxic and pro-apoptotic effects in co-coltures of cardiomyocytes and hPBMC. OxLDL increases significantly the nivolumab-induced cardiotoxicity in a manner that is sensitive to TLR4 and NLRP3. Incubation of cardiomyocytes with ox-LDL (10 and 50 μg/mL) for 24 hours increased TLR4 and NF-κB expressions. Ox-LDL had pro-apoptotic effects in a concentration-dependent manner with the involvement of lipid peroxidation but not of intracellular calcium. Conclusion Ox-LDL exacerbates cardiotoxicity during exposure to nivolumab through pro-inflammatory mechanisms. These results place the first step to preclinical studies aimed to reduce ox-LDL during treatment with ICIs through pharmacological inhibition or by changing diet and lifestyle. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministero della Salute, Ricerca Corrente project

Anthracyclines are essential adjuvant therapies for a variety of cancers, particularly breast, gastric and esophageal cancers. Whilst prolonging cancer-related survival, these agents can induce drug-related cardiotoxicity. Spirulina, Reishi (Ganoderma Lucidum) and Moringa are three nutraceuticals with anti-inflammatory effects that are currently used in cancer patients as complementary and alternative medicines to improve quality of life and fatigue. We hypothesize that the nutraceutical combination of Spirulina, Reishi and Moringa (Singo) could reduce inflammation and cardiotoxicity induced by anthracyclines. Female C57Bl/6 mice were untreated (Sham, n = 6) or treated for 7 days with short-term doxorubicin (DOXO, n = 6) or Singo (Singo, n = 6), or pre-treated with Singo for 3 days and associated with DOXO for remaining 7 days (DOXO–Singo, n = 6). The ejection fraction and radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100, Fujifilm). The myocardial expressions of NLRP3, DAMPs (galectin-3 and calgranulin S100) and 13 cytokines were quantified through selective mouse ELISA methods. Myocardial fibrosis, necrosis and hypertrophy were analyzed through immunohistochemistry (IHC). Human cardiomyocytes were exposed to DOXO (200 nM) alone or in combination with Singo (at 10, 25 and 50 µg/mL) for 24 and 48 h. Cell viability and inflammation studies were also performed. In preclinical models, Singo significantly improved ejection fraction and fractional shortening. Reduced expressions of myocardial NLRP3 and NF-kB levels in cardiac tissues were seen in DOXO–Singo mice vs. DOXO (p < 0.05). The myocardial levels of calgranulin S100 and galectin-3 were strongly reduced in DOXO–Singo mice vs. DOXO (p < 0.05). Immunohistochemistry analysis indicates that Singo reduces fibrosis and hypertrophy in the myocardial tissues of mice during exposure to DOXO. In conclusion, in the preclinical model of DOXO-induced cardiotoxicity, Singo is able to improve cardiac function and reduce biomarkers involved in heart failure and fibrosis.

Background The clinical trial “DECLARE-TIMI 58” (Dapagliflozin Effect on Cardiovascular Events-Thrombolysis in Myocardial Infarction 58), demonstrated that dapagliflozin, a Sodium glucose cotransporter 2 inhibitor, reduces the composite end point of cardiovascular death/hospitalization for heart failure in a broad population of patients with type 2 diabetes mellitus. Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, achieving unprecedented efficacy in multiple malignancies. However, ICIs are associated with immune-related adverse events involving cardiotoxicity. Purpose We aimed to study if dapagliflozin could affect ipilimumab-induced anticancer efficacy in human breast cancer cells and reduces its cardiotoxicity. Methods Co-culture of hPBMCs and human cardiomyocytes or estrogen-responsive and triple-negative breast cancer cells (MCF-7 and MDA-MB-231 cell lines) were exposed to ipilimumab (200 nM) alone or combined to SGLT-2 inhibitor (dapagliflozin) for 72h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of TLR4/MyD88; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Results Dapagliflozin increases significantly the cardiomyocytes viability during exposure to Ipilimumab. Indeed, in human breast cancer cells, dapagloflozin showed an opposite behavior with a significant increase in cell mortality and apoptosis (p&lt;0.001 vs only ipilimumab). Cardioprotective properties of dapagliflozin are explainable by the reduction of intracellular Ca2+ overload (−56,8% vs only ipilimumab; p&lt;0,001), of the lipid peroxidation (mean reduction of 42,1–48,6% compared to cells exposed only to ipilimumab; p&lt;0,05). Moreover, cells exposed to dapagliflozin during ipilimumab reduced the protein expression of pro-inflammatory cytokines involved in cardiotoxicity and resistance to anticancer effects of ICIs (−47,2% for Interleukin-1β; −48,7 for Interleukin 6; −32,1% for Interleukin 8; p&lt;0,001 for all vs only ipilimumab groups). Notably, dapagliflozin reduces p65-NF-κB activation (−46,3 and −49,3% vs only ipilimumab, p&lt;0.05) and inhibits of 43,2–53,7% the expression of NLRP3 inflammasome, p&lt;0.05 for all). No significant effects were seen on TLR4/MYD88 expression in all groups. Conclusion Dapagliflozin demonstrated cardioprotective properties during Ipilimumab exposure in co-culture model of hPBMCs and cardiomyocytes. Dapagliflozin improves Ca2+ homeostasis and inhibits the pro-inflammatory “NLRP3–NF-κB–cytokines” pathways in cardiac cells. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ricerca corrente grant of Italian Ministry of Health

e15039 Background: Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel therapy to treat hypercholesterolaemia and related cardiovascular diseases. Evolocumab, a PCSK9 inhibitor, reduced the risk of cardiovascular events in patients with atherosclerotic cardiovascular diseases when added to maximally tolerated statin therapy (± ezetimibe), and recent data from the ODYSSEY OUTCOMES trial indicate that alirocumab added to maximally tolerated statin therapy (± other lipid-lowering drugs) reduces the risk of cardiovascular events in patients with a recent acute coronary syndrome. Methods: Human fetal cardiomyocytes (HFC cell line), human HER2+ breast cancer cells were exposed to subclinical concentration of doxorubicin, trastuzumab, sequential treatment of both (all 100 nM), alone or in combination with evolocumab (50 nM) for 24 and 48h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of TLR4/MyD88; mTORC1 Fox01/3a; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Results: Evolocumab co-incubated with doxorubicin alone or in sequence with trastuzumab exerts cardioprotective effects, enhancing cell viability of 35-43% compared to untreated cells (p < 0,05 for all); in cardiomyocytes Evolocumab reduced significantly the cardiotoxicity through MyD88/NF-KB/cytokines axis and mTORC1 Fox01/3α mediated mechanisms. In human HER2+ breast cancer cells, co-exposure of Evolocumab with doxorubicin and trastuzumab increased significantly cell apoptosis and necrosis through the involvement of key cytokines involved in chemoresistence. Conclusions: We demonstrated, for the first time, that the PCSK9 inhibitor evolocumab exerts direct effects in cardiomyocytes and human HER2+ breast cancer cells during doxorubicin and trastuzumab exposure turning on a new light on its possible use in the management of breast cancer therapies.

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