Briefly, human cardiac cells, called HFCs (Innoprot, Derio, Spain), were cultured in a selective culture medium + Fetal Bovine Serum (FBS 10%v/V, Sigma Aldrich, St. Louis MO, USA), Penicillin at 50 U/ml, Streptomycin at 50 μg/ml and L-Glutamine at 1%v/V. Cardiomyocytes were than plated in a 96 well plates (10,000 cells/well) for 16 h. Human Peripheral Blood Mononuclear Cells (hPBMCs) were added at effector: target ratio of 5:1 with and without ICIs in monotherapy or combined regimen, such as Relatlimab or Nivolumab (or both combined) and Atezolizumab or Ipilimumab (or both combined), all used at 100 nM and incubated for 48 h at 37 °C, as described in other experimental research (19, 20) (Figure 1). ICIs molar concentration reflects the dose reached in patient serum/plasma administered with the conventional therapeutic doses of immunomodulatory mAbs (10 mg/kg) (23). Control groups were identified as cells untreated or treated with a standard IgG. Notably, experimental conditions including ratio of cellular components are in line with recent established work of our and other group that mimic lymphocyte-cardiomyocyte interaction in preclinical and clinical models (19, 20, 23).

As demonstrated in our previous work (23), considering the expression of PD-L1, PD-1 and CTLA-4 on cardiomyocytes, after treatment with antibodies described in paragraph 2.1, lymphocytes were removed (collecting the supernatant of co cultures). Cardiac cell lysis was determined through the quantification of released LDH (LDH detection kit, Thermo-Fisher Scientific, Meridian Rd., Rockoford, IL, USA), following the manufacturer's instructions. The sensitivity of this method was below 0.01 OD for absorbance at 490 and 680 nm, and the assay accurately detected LDH release in the range of 0.01–0.4 OD. Reported values refer to a sample of cells exposed exclusively to Triton × 100 lysis solution at 10%v/V.

Histone deacetylases (HDACs) type 4 are enzymes that affect gene expression through acetylation of core histones of chromatin and are involved in pathogenesis of chemotherapy-induced heart failure (24, 25). After treatments described in paragraph 2.1, cardiomyocytes were harvested and lysed through a lysis buffer (1 mM EDTA, 20 mM NaF, 3 mM Na₃VO₄, 100 mM NaCl, 1 mM PMSF, 50 mM Tris/HCl, selective protease inhibitor). Cell lysates were centrifuged and supernatants were analyzed through Human HDAC4 ELISA Kit (AB300311, AbCam, Milan Italy) in line with manufacturer's instructions. Control group refers only to untreated cells (Untreated HFC); sensitivity of the ELISA was 94.776 pg/ml and the assay accurately detected HDAC4 expression in the range of 312.5–20,000 pg/ml).

Intracellular Ca⁺⁺, ATP content and NADH:ubiquinone oxidoreductase core subunit S expression reflect the mitochondrial activity of cardiomyocytes and their metabolic integrity (26–28). For intracellular calcium quantification, a fluorescence dye Fluo-3 AM, following the manufacturer's protocol, was used. In brief, after treatments described in paragraph 2.1, cardiomyocytes were loaded with 5 μM Fluo-3 AM at 37 °C for 30 min in the dark, and then washed three times with PBS to remove excess of dye. The fluorescence intensity of Fluo-3 chelated with calcium was recorded on a microplate spectrofluorometer (xMark Microplate, Spectrofluorometer Biorad, Milan, Italy) at excitation and emission wavelengths of 488 and 525 nm, respectively. For ATP and Ndufs1 analysis, after treatments described in paragraph 2.1, cardiomyocytes were harvested and lysed through a lysis buffer (1 mM EDTA, 20 mM NaF, 3 mM Na₃VO₄, 100 mM NaCl, 1 mM PMSF, 50 mM Tris/HCl, selective protease inhibitor). Cell lysates were centrifuged and supernatants were analyzed through ATP Assay Kit (Colorimetric/Fluorometric) (AB83355, AbCam, Milan, Italy) and Human NADH Dehydrogenase Ubiquinone Fe-S Protein 1 (NDUFS1) ELISA Kit (Abbexa, Uk) in line with manufacturer's instructions. Control group refers only to untreated cells (Untreated HFC); sensitivity of the ATP assay was <1 µM. Sensitivity of the ELISA was <0.12 ng/ml and the assay accurately detected Ndufs1 expression in the range of 0.312 ng/ml–20 ng/ml).

Intracellular reactive oxygen species and lipid peroxidation are key mediators of anticancer drug-induced cardiovascular diseases, including myocarditis (29). In brief, after treatments described in paragraph 2.1, cardiomyocytes were washed three times with cold PBS, harvested with 0.25%v/v Trypsin and centrifuged at 1,000×g for 10 min. The supernatant was discarded and the cell pellet sonicated in cold PBS. After a centrifugation step at 800×g for 5 min, we quantified intracellular ROS and two markers of lipid peroxidation called malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNA) through commercial kit with a spectrophotometer according to the manufacturer's protocols (Sigma Aldrich, Milan, Italy). According to the kit instructions, the total protein content of cardiac cell homogenates was quantified through Micro BCA protein assay kit (Pierce, Thermo Fisher, Milan, Italy).

NLRP-3 (inflammasome) is the key driver of cardiovascular diseases and anticancer-drug induced cardiotoxicity (30); therefore, after treatments described in paragraph 2.1, cells were harvested and lysed through a lysis buffer (1 mM EDTA, 20 mM NaF, 3 mM Na₃VO₄, 100 mM NaCl, 1 mM PMSF, 50 mM Tris/HCl, selective protease inhibitor). Cell lysates were centrifuged and supernatants were analyzed through NLRP-3 ELISA Kit (code OKEH03368; Aviva Systems Biology, San Diego, CA, USA) in line with manufacturer's instructions (31). Control group refers only to untreated cells (Untreated HFC); sensitivity of the ELISA was 0.078 ng/ml and the assay accurately detected NLRP-3 expression in the range of 0.1–40 ng/ml).

Pro-inflammatory cytokines are key players of heart failure and myocarditis (32, 33). As recently described in literature (33) were quantified in supernatant of cardiac cells exposed to combined ICIs therapies through ELISA methods. Briefly, after treatment with Relatlimab or Nivolumab (or both combined) and Atezolizumab or Ipilimumab (or both combined), culture supernatants were centrifuged and analyzed for IL-1β, IL-6, TNF-α, IL-4, IL-23, IL-17a, IL-12, IL-18 and INF-γ quantification through ELISA method in line with literature (31) (Sigma Aldrich, Milan, Italy) in line with manufacturer's instructions (31). Control group refers only to untreated cells (Untreated HFC). In order to confirm whether the release of cytokines is from cardiomyocytes or lymphocytes, cytokine release tests were also performed in in vitro culture models of HFCs only and lymphocytes only (results are available in Supplementary Figure 1). Sensitivity of IL-1β Elisa kit: 0.3 pg/ml and refers to a standard curve range: 0.48–500 pg/ml of IL-1β. Sensitivity of IL-6 Elisa kit: 3 pg/ml and refers to a standard curve range: 1.37–1,000 pg/ml. Sensitivity of TNF-α Elisa kit: 30 pg/ml and refers to a standard curve range: 1,56–1,000 pg/ml. Sensitivity of INF-γ Elisa kit: 15 pg/ml and refers to a standard curve range: 20.6–15,000 pg/ml. Sensitivity of IL-4 Elisa kit: 5 pg/ml and refers to a standard curve range: 3.3–200 pg/ml. Sensitivity of IL-23 Elisa kit: 15 pg/ml and refers to a standard curve range: 28.6–7,000 pg/ml. Sensitivity of IL-17 Elisa kit: 0.5 pg/ml and refers to a standard curve range: 1.6–150 pg/ml. Sensitivity of IL-12 Elisa kit: 1 pg/ml and refers to a standard curve range: 0.8–600 pg/ml. Sensitivity of IL-18 Elisa kit: 20 pg/ml and refers to a standard curve range: 24.7–18,000 pg/ml.

The heart-type fatty acid binding protein (called H-FABP) is a new putative biomarker of cardiotoxicity (29, 34, 35); in fact, after 30 min of myocardial injury, plasma levels of hFABP are significantly increased (36). After treatments described in paragraph 2.1, cells were harvested and lysed as described in paragraph 2.3. Intracellular levels of H-FABP (AbCam, Cambridge, UK cat: ab243682) were measured through ELISA method in line with literature (37) and manufacturer's instructions. Control group refers only to untreated cells (Untreated HFC). Sensitivity of H-FABP Elisa kit: 2.4 pg/ml and refers to a standard curve range: 10.9–700 pg/ml. The NT-pro-brain natriuretic peptide (NT-proBNP), troponin T and BNP are conventional biomarkers of heart failure and recently included in cardiac monitoring of ICIs-mediated cardiovascular diseases (38, 39). To test and confirm if combinatorial ICIs therapies could induce cardiac damages in lymphocyte-cardiomyocyte models, after treatment with Relatlimab or Nivolumab (or both combined) and Atezolizumab or Ipilimumab (or both combined) cardiomyocytes were tested for NT-proBNP production through Human NT-proBNP ELISA Kit (AbCam, Cambridge, UK cat: ab263877), Troponin T (TNNT1) Human ELISA Kit (EHTNNT1,Thermo Fisher, Milan, Italy), and human BNP ELISA Kit (EHNPPB, Thermo Fisher, Milan, Italy) in line with manufacturer's instructions. Control group refers only to untreated cells (Untreated HFC). Sensitivity of NT-Pro-BNP Elisa kit, Troponin T and BNP: 11.5 pg/ml, 0.35 ng/ml and 14 pg/ml, respectively, and refers to a standard curve range: 21.9–1,400 pg/ml.

All analysis were performed in triplicates (n = 3). Results were presented as mean ± standard deviation (SD). A paired-t test was used to compare different treatments, by using a Sigmaplot software (Systat Software Inc., San Jose, CA, USA). p < 0.05 was considered as statistically significant difference between groups.

To mimic the lymphocyte infiltration that is clinically found in cancer patients treated with ICIs who develop myocarditis, we have developed a cellular model of lymphocyte-cardiomyocyte interaction, already described in previous works (19, 22, 40) (Figure 1). To this aim, human cardiomyocyte/hPBMCs co-culture was exposed or unexposed to 100 nM Relatlimab, Nivolumab, Atezolizumab, Ipilimumab or their combinations as used in clinical trials or approved by FDA. We found that PD-L1 and CTLA-4 or PD-1 and LAG-3 blocking agents induced a higher cardiac cell lysis than the single agent treatments (see Figure 2). In fact, cells exposed to Relatlimab, Nivolumab and both in combination had a percentage of LDH release of 14 ± 3.6, 32 ± 2.1 and 59 ± 3.6%, respectively, vs. Unrelated IgG group (control; 8% ± 3.1; p < 0.001). Similarly, cells exposed to Atezolizumab, Ipilimumab and both in combination had a percentage of LDH release 18 ± 3.9, 30 ± 3.4 and 43 ± 5.2%, respectively, vs. Unrelated IgG group (control; 15% ± 4.0; p < 0.001).

To clarify whether this enhanced effect was due to a higher activation of cytotoxic immune cells, we analyzed the levels granzyme B in co-culture supernatant and we found that the two combinations induced higher secretion of the cytokines, markers of T cell activation, than single agent treatments, in line with the formulated hypothesis. In details, co-cultures exposed to Relatlimab, Nivolumab and both in combination led to a granzyme B release of 3,850 ± 8, 4,705 ± 28, 10,075 ± 53% vs. Unrelated IgG group (control; 4,087 ± 16; p < 0.001). Similarly, cells exposed to Atezolizumab, Ipilimumab and both in combination led to a Granzyme B release of 4,750 ± 25%, 9,885 ± 34%, 12,265 ± 59% vs. Unrelated IgG group (control, 4,182,5 ± 2,66%; p < 0.001). Notably, Atezolizumab + Ipilimumab does not show statistically significant differences to Ipilimumab (p > 0.05). Moreover, as reported in Figure 3, cardiomyocytes co-cultured with hPBMC and incubated with ICIs change their cellular morphology. Some cell debris and a significant reduction in their fibroblast-like phenotype is seen, indicating cellular side effects of ICIs therapies.

Histone deacetylases (HDACs) are enzymes that play a critical role in cardiac function and ischemic injury (41). Patients with heart failure and atherosclerosis have high levels of HDAC-4 expression (42). Myocardial ischemic injury resulted in increases in HDAC4 protein levels. Therefore we investigated on the effect of ICIs therapy on HDAC-4 expression in HFC cell lysates. Ipilimumab, Atezolizumab, Relatlimab and Nivolumab (in monotherapy and combinatorial therapy regimens) increased protein levels of HDAC-4 in HFC cells (Figure 4). In detail, untreated HFC cells co-cultured with hPBMC, had a HDCAC-4 expression of 336.3 ± 32.3 pg/ml. Instead, Atezolizumab, Ipilimumab and both in combination increased HDAC-4 protein levels significantly (533.6 ± 25.7; 488.3 ± 28.2; 771.1 ± 33.7 pg/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control). Nivolumab, Relatlimab and both in combination increased significantly HDAC-4 expression compared to HFC/hPBMC group (456.4 ± 35.1; 508.8 ± 28.5; 733.7 ± 33.8 pg/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control). This behavior is in line with other preclinical work that highlight the HDAC-4 overexpression in cardiomyocytes exposed to cardiotoxic drugs, including anthracyclines.

To study the effects of ICIs therapies on mitochondrial functions and metabolic activity of cardiomyocytes, intracellular Ca⁺⁺ concentration, ATP content and NADH:ubiquinone oxidoreductase core subunit S1 (Ndufs1) levels were quantified. Reduced Ndufs1 levels were associated to mitochondrial dysfunctions through the reduction of ATP production in cardiomyocytes, resulting in cardiac dysfunction (36, 43). Immune checkpoint inhibitors increased intracellular calcium content in HFC lysates, indicating changes of the contractile balance of cardiomyocytes, in line with the literature data of the same cells treated with anthracyclines, trastuzumab and other cardiotoxic drugs. Combinatorial ICIs therapy significantly increases intracellular calcium concentration than monotherapy (p < 0.001) (Figure 5). Specifically, untreated HFC cells co-cultured with hPBMC, had an intracellular Ca⁺⁺ content of 44.7 ± 9.6 a.u. Instead, Atezolizumab, Ipilimumab and both in combination increased intracellular Ca⁺⁺ levels significantly (112.2 ± 16.4; 120.6 ± 21.2; 277.7 ± 17.7 a.u for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control). Nivolumab, Relatlimab and both in combination increased significantly the intracellular Ca⁺⁺ levels compared to untreated HFC/hPBMC group (163.3 ± 21.1; 188.5 ± 18.6; 321.2 ± 22.4 a.u for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control). ATP production and Ndufs1 expression are significantly changed in HFCs exposed to ICIs, indicating mitochondrial damage induced by lymphocyte activation against cardiomyocytes under exposure to ICIs in monotherapy and combinatorial regimens (Figure 5). ATP content was drastically and significantly reduced after exposure with ICIs therapy and the same behavior was seen on Ndufs1. In detail, ATP content of untreated HFC cells co-cultured with hPBMC, was 33.2 ± 4.1 µM; instead, Atezolizumab, Ipilimumab and both in combination decreased significantly ATP content (12.2 ± 2.2, 11.4 ± 2.7, 6.5 ± 2.1 µM for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control). Nivolumab, Relatlimab and both in combination decreased significantly ATP levels compared to untreated HFC/hPBMC group (15.2 ± 4.1, 13.3 ± 2.7, 8.8 ± 2.9 µM for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control). Moreover, Ndufs1 protein levels of untreated HFC cells co-cultured with hPBMC, was 15.9 ± 1.3 ng/ml; Atezolizumab, Ipilimumab and both in combination decreased significantly Ndufs1 expression (10.3 ± 2.5, 9.4 ± 2.2, 5.9 ± 1.8 ng/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control). Nivolumab, Relatlimab and both in combination decreased significantly the Ndufs1 levels compared to untreated HFC/hPBMC group (9.6 ± 2.7, 10.1 ± 2.1, 4.6 ± 1.7 ng/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control).

Intracellular Oxygen Species and lipid peroxidation are strictly involved in anticancer drug-mediated cardiotoxicity, including immune-mediated cardiovascular affections (19, 20). In human cardiomyocytes co-cultured with hPBMC, ICIs in monotherapy and combinatorial regimen increased significantly iROS and lipid peroxidation products MDA and 4-HNA (Figure 6). In detail, fluorescence intensity related to iROS levels in cell lysates of untreated HFC cells, co-cultured with hPBMC, was 34.4 ± 6.6 a.u; instead, Atezolizumab, Ipilimumab and both in combination increased significantly I ROS levels (73.2 ± 10.3, 65.5 ± 12.2, 112.2 ± 9.5 a.u for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control). Nivolumab, Relatlimab and both in combinatorial regimen increased fluorescence intensity related to iROS content compared to HFC/hPBMC group (59.8 ± 8.8, 66.1 ± 7.5, 125.7 ± 14.3 a.u for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control). MDA and 4-HNA levels in cell lysates of untreated HFC cells co-cultured with hPBMC, were 0.71 ± 0.17 and 0.64 ± 0.3 nmol/ml, respectively. Instead, Atezolizumab, Ipilimumab and both in combination increased drastically their levels (1.2 ± 0.12, 1.33 ± 0.2, 3.2 ± 0.23 nmol/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control for MDA; 1.2 ± 0.12, 0.97 ± 0.26, 2.8 ± 0.32 nmol/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control for 4-HNA). The same behavior was seen for Nivolumab, Relatlimab and both in combinatorial regimen, with a significant increase in MDA and 4-HNA levels, compared to untreated HFC/hPBMC group (0.9 ± 0.18, 0.83 ± 0.23, 2.9 ± 0.3 nmol/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control for MDA; 0.99 ± 0.22, 1.3 ± 0.23, 3.1 ± 0.35 nmol/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control for 4-HNA) (Figure 6).

Therapies based on ICIs are associated to cardiac and vascular injuries that correlate with high hsCRP levels indicating systemic inflammation (44). Analyses of protein expression through ELISA method in hPBMC –cardiomyocyte models exhibited highly significant increases in NLRP-3 induced by Atezolizumab, Ipilimumab, Nivolumab and Relatlimab in monotherapy compared to untreated HFC and untreated HFC-hPBMC co-cultures (Figure 7). Interestingly, combinatorial ICIs therapies increased of five times the NLRP3 expression (p < 0.001 vs. monotherapy). In detail, NLRP3 protein levels in cell lysates of untreated HFC cells, co-cultured with hPBMC, were 4.7 ± 2.1 pg/ml. Instead, Atezolizumab, Ipilimumab and both in combination increased drastically NLRP3 levels (16.6 ± 1.6, 17.3 ± 1.8, 26.2 ± 1.7 pg/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control). Nivolumab, Relatlimab and both in combinatorial regimen drastically increased NLRP3 protein levels than untreated HFC/hPBMC group (16.4 ± 2.1, 17.7 ± 2.5, 24.3 ± 1.8 pg/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control). Considering that NLRP-3 drives cytokine storm (45), it was evaluated if ICIs therapies could enhance IL-6 and IL-1β levels. In line with NLRP-3 release results, ICIs monotherapy and especially combinatorial therapies strongly enhanced both cytokines, indicating a pro-inflammatory phenotype. In detail, IL-1β and IL-6 protein levels in supernatant of untreated HFC cells co-cultured with hPBMC, were 75.6 ± 12.2 and 47.8 ± 11.1 pg/ml, respectively. Instead, Atezolizumab, Ipilimumab and both in combination increased drastically their levels (188.5 ± 14.2, 332.4 ± 17.2, 471.7 ± 13.6 pg/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control for IL-1β;122.2 ± 13.1, 228.6 ± 14.5, 396.7 ± 12.8 pg/mL for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control for IL-6). The same behavior was seen for Nivolumab, Relatlimab and both in combinatorial regimen, with a significant increase in IL-1β and IL-6 level, compared to untreated HFC/hPBMC group (134.2 ± 13.7, 177.2 ± 17.4, 383.5 ± 16.3 pg/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control for IL-1β; 89.6 ± 10.6, 114.8 ± 11.5, 277.8 ± 12.3 pg/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control for IL-6) (Figure 7).

Based on Tarhini et al. results (46) that ICIs increases circulating pro-inflammatory cytokines like tumor necrosis factor (TNF)-α and interferon (IFN)-γ, could induce a similar behavior in lymphocyte-cardiomyocyte models. In line with clinical data of Tarhini et al., it was found that Atezolizumab, Ipilimumab, Nivolumab and Relatlimab in monotherapy increased interferon (IFN)-γ and tumor necrosis factor (TNF)-α levels in cardiomyocytes. Evidences of synergistic pro-inflammatory effects of combinatorial ICIs regimen compared to monotherapies are seen. In details, (IFN)-γ and (TNF)-α protein levels in supernatant of untreated HFC cells co-cultured with hPBMC, was 11.2 ± 5.2 and 18.5 ± 8.55 pg/ml, respectively. Instead, Atezolizumab, Ipilimumab and both in combination increased drastically their release (32.6 ± 4.38, 42.3 ± 4.0, 63.5 ± 6.74 pg/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control for IFN-γ; 74.3 ± 9.43, 65.7 ± 8.6, 97.5 ± 7.9 pg/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab group, respectively; p < 0.001 vs. control for TNF-α). The same behavior was seen for Nivolumab, Relatlimab and both in combinatorial regimen, with a significant increase in IFN-γ and TNF-α release than untreated HFC/hPBMC group (26.7 ± 3.67, 31.2 ± 4.22, 55.8 ± 6.8 pg/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control, relatively to IFN-γ; 57.5 ± 6.3,63.2 ± 5.77, 89.4 ± 7.32 pg/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab group, respectively; p < 0.001 vs. control, relatively to TNF-α) (Figure 7). The same behavior was seen for other pro-inflammatory cytokines involved in ICIs-mediated myocardial affections, such as IL-4, IL-23, IL-17a, IL-12 and IL-18 that, in line with literature (33), are significantly enhanced after ICIs therapy due to the induced immune-mediated pro-inflammatory phenotype.

Patients with high levels of H-FABP are exposed to increased risk of death and cardiomyopathies; Troponin T, BNP and NT-pro-BNP are established biomarkers of cardiotoxicity induced by anthracyclines, immune checkpoint inhibitors and radiotherapy (47, 48). Therefore, it was studied if combined immune checkpoint blockade could increase the secretion of H-FABP, Troponin-T, BNP and NT-pro-BNP form cardiac cells (Figure 8). First, Atezolizumab, Ipilimumab, Nivolumab and Relatlimab in monotherapy were able to increase significantly all biomarkers of cardiotoxicity; specifically, H-FABP levels were 23.2 ± 3.2, 32.1 ± 2.6 and 62.1 ± 3.3 pg/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab groups, respectively; p < 0.001 vs. control; 18.8 ± 3.5, 22.8 ± 3.2, 58.8 ± 4.1 pg/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab groups, respectively; p < 0.001 vs. control. Moreover, NT-pro-BNP levels were 146.5 ± 10.4, 165.4 ± 17.3 and 188.6 ± 15.9 pg/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab groups, respectively; p < 0.001 vs. control; 132.2 ± 11.2, 143.7 ± 13.2 and 178.5 ± 17.8 pg/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab groups, respectively; p < 0.001 vs. control.Therefore, also in this case, Atezolizumab associated to Ipilimumab and Nivolumab associated to Relatlimab exerted synergic cardiotoxic properties with a significant enhancement of NT-proBNP and H-FABP compared to monotherapies (Figure 8).

The same behavior was seen for Troponin-T and BNP: in brief, Atezolizumab, Ipilimumab, Nivolumab and Relatlimab in monotherapy were able to increase significantly all biomarkers of cardiotoxicity; specifically, Troponin-T levels were 0,48 ± 0.04, 0.37 ± 0.037 and 0.77 ± 0.023 ng/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab groups, respectively; p < 0.001 vs. control; 0,26 ± 0.025, 0.34 ± 0.04 and 0.69 ± 0.045 ng/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab groups, respectively; p < 0.001 vs. control. Moreover, BNP levels were 53.7 ± 8.4, 61.1 ± 9.3 and 92.1 ± 6.6 pg/ml for Atezolizumab, Ipilimumab and Atezolizumab/Ipilimumab groups, respectively; p < 0.001 vs. control; 41.2 ± 7.3, 50.4 ± 3.7 and 72.2 ± 12.1 pg/ml for Nivolumab, Relatlimab and Nivolumab/Relatlimab groups, respectively; p < 0.001 vs. control (Figure 8).