Schematic drawing of mechanisms operated by ZA to reverse chemoresistance and immune-resistance.

<p><b>A</b>. The accelerated Mev pathway in MDR+ cells leads to the constitutive activation of Ras/ERK1-2 and RhoA/RhoA kinase downstream signalling pathways which culminates into HIF-1α activation and plasma membrane Pgp expression. The higher amounts of plasma membrane-associated cholesterol in MDR+ cells also contribute to the functional Pgp activation. The higher efficiency to extrude Dox protects MDR+ cells from cytotoxicity and ICD epitomized by CRT exposure on the cell surface. <b>B</b>. By inhibiting the Mev pathway, ZA downregulates the Ras/ERK1-2 and RhoA/RhoA kinase signalling pathways, and decreases the HIF-1α-induced transcription of Pgp. As a result, Dox accumulates inside MDR+ cells at sufficient amounts to induce cytotoxicity and promote CRT exposure, turning the phenotype of these cells from a chemoimmunoresistant phenotype into a chemoimmunosensitive phenotype.</p

Correlation between intracellular doxorubicin retention, <i>mdr1</i> expression and Mev pathway activity in MDR− and MDR+ tumor cells.

<p><b>A</b>. Intracellular doxorubicin (Dox) concentrations in MDR− cells (HT29, A549 and MCF7), in the corresponding acquired MDR+ counterparts (HT29-dx cells, A549-dx cells, MCF7-dx), and constitutive MDR+ cells (HepG2, HP06, HMM). Significantly lower concentrations were detected in cells with acquired MDR <i>vs</i> MDR− cells (HT29-dx <i>vs</i> HT29: *p<0.002; A549-dx <i>vs</i> A549: *p<0.001; MCF7-dx <i>vs</i> MCF7: *p<0.001), and in cells with constitutive MDR <i>vs</i> MDR− cells (mean value of intracellular doxorubicin in HepG2/HP06/HMM <i>vs</i> mean value in HT29/A549/MCF7: °p<0.001). <b>B</b>. <i>mdr1</i> mRNA expression. Significant higher <i>mdr1</i> levels were observed in cells with acquired MDR <i>vs</i> MDR− cells (HT29-dx <i>vs</i> HT29: *p<0.002; A549-dx <i>vs</i> A549: *p<0.002; MCF7-dx <i>vs</i> MCF7: *p<0.001), and in cells with constitutive MDR <i>vs</i> MDR− cells (mean value of <i>mdr1</i> levels in HepG2/HP06/HMM <i>vs</i> mean value in HT29/A549/MCF7: °p<0.001). <b>C</b>. Rate of cholesterol synthesis. Significant higher activity was measured in cells with acquired MDR <i>vs</i> MDR− cells (HT29-dx <i>vs</i> HT29: *p<0.002; A549-dx <i>vs</i> A549: *p<0.002; MCF7-dx <i>vs</i> MCF7: *p<0.002), and in cells with constitutive MDR <i>vs</i> MDR− cells (mean value of cholesterol synthesis in HepG2/HP06/HMM vs mean value in HepG2/HP06/HMM: °p<0.001). <b>D</b>. Direct correlation between the rate of cholesterol synthesis and the expression levels of <i>mdr1</i> in individual cell lines (r² = 0.95). For panels <b>A</b>, <b>B</b>, and <b>C</b> bars represent the mean ± SD of 3 independent experiments.</p

ZA increases the internalization of MDR+ cells by autologous DCs and the subsequent activation of cytotoxic CD8+ T cells.

<p><b>A</b>. DC-mediated internalization of HT29, HT29-dx, and HMM cells after incubation with ZA and/or Dox. Tumor cells were incubated for 48 h without (CTRL) or with 1 µmol/L ZA, for 24 h with 1 µmol/L Dox, for 48 h with 1 µmol/L ZA, followed by 1 µmol/L Dox for additional 24 h (ZA+Dox). Dox alone and ZA+Dox significantly increased internalization of HT29 cells (*p<0.02). ZA+Dox increased internalization of HT29-dx (°p<0.005) and HMM (^◊p<0.005) cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060975#s3" target="_blank">Results</a> represent the mean ± SD of 3 independent experiments. <b>B</b>. Fluorescence microscopy analysis of HT29-dx internalization after 6 and 24 h incubation with DCs. HT29-dx cells were incubated with ZA+Dox as reported in A. Micrographs are from one representative of 3 experiments. <b>C</b>. Cytotoxic activation of CD8+ T cells after 10 days incubation of purified T cells with autologous DCs pulsed with HT29-dx tumor cells, treated as reported in a. Cytofluorometric analysis of cell surface CD107 expression was used as a marker of specific TCR-induced CD8+ T-cell degranulation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060975#s3" target="_blank">Results</a> are from one representative of 4 experiments. <b>D</b>. Pooled data of CD107 expression on CD8+ T cells after incubation with autologous DC as reported above. Bars represent the mean ± SEM of 4 experiments.</p

Effects of ZA and inhibitors of ERK1/2, RhoA kinase, HIF-1α on MDR+ cells.

<p><b>A</b>. Phospho(Ser)-HIF-1α (pHIF-1α) and total HIF-1α expression in HMM cells left untreated (CTRL) or treated for 24 h at 10 µmol/L with the ERK1/2 kinase inhibitor PD98059 (PD), RhoA kinase inhibitor Y27632 (Y27), HIF-1α inhibitor YC-1 (YC), and 1 µmol/L ZA for 48 h. GAPDH data are shown to confirm equivalent per lane protein loading. <b>B</b>. HIF-1 activity in HMM cells left untreated (CTRL) or treated as reported in panel <b>A</b>. All differences between treated <i>vs</i> untreated cells are statistically significant (* p<0.01). <b>C</b>. Pgp expression in HMM cells of untreated (CTRL) and treated as reported in panel <b>A</b>. <b>D</b>. Intracellular doxorubicin concentrations in HMM cells in cells incubated as reported above in medium alone (CTRL), followed by 1 µmol/L Dox for a further 24 h. Differences between treated <i>vs</i> untreated cells are statistically significant (*p<0.01). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060975#s3" target="_blank">Results</a> shown in panels <b>A</b> and <b>C</b> are representative data from one of 2 experiments. For panels <b>B</b> and <b>D</b>, the results represent the mean ± SD of 3 independent experiments.</p

Effects of ZA on cholesterol and isoprenoid synthesis, Ras/RhoA isoprenylation, and ERK1/2 and RhoA kinase activity in MDR− and MDR+ cancer cells.

<p>MDR− HT29, and MDR+ HT29-dx and HMM cells were cultured without (CTRL) or with zoledronic acid (ZA). For panels <b>B–E</b>, ZA (1 µmol/L) was used for 48 h, FTI-277 (10 µmol/L, FTI), GGTI-286 (10 µmol/L, GGTI), Y27632 (10 µmol/L, Y276) for 24 h. <b>A</b>. <i>Left panel</i>: dose-dependent inhibition of cholesterol synthesis in cells treated with 0.01–10 µmol/L ZA for 24 h. Inhibition was statistically significant in HT29 (*p<0.001), HT29-dx (°p<0.01) d HMM cells (^◊p<0.005) <i>vs</i> baseline values (0). <i>Right panel</i>: time-dependent inhibition of cholesterol synthesis in cells treated with 1 µmol/L ZA for 24–72 h. Inhibition was statistically significant in HT29 (*p<0.001), HT29-dx (°p<0.0001) and HMM cells (^◊p<0.001) <i>vs</i> baseline values (0). For both panels: HT29-dx/HMM <i>vs</i> HT29: *p<0.001. <b>B</b>. MDR+ cells synthesized higher amounts of FPP (<i>left panel</i>) and GGPP (<i>right panel</i>) than MDR− cells (*p<0.005). ZA significantly lowered FPP synthesis <i>vs</i> untreated (CTRL) cells (HT29: *p<0.001; HT29-dx: °p<0.002; HMM: ^◊p<0.001) and GGPP synthesis <i>vs</i> untreated (CTRL) cells (HT29:*p<0.02; HT29-dx: °p<0.001; HMM:^◊p<0.005). <b>C</b>. MDR+ cells displayed an unbalanced distribution between isoprenylated membrane-bound (<i>M</i>) and non isoprenylated cytosolic (<i>C</i>) Ras (<i>left panel</i>) and RhoA (<i>right panel</i>) compared with MDR− cells. ZA treatment increased the amount of cytosolic Ras and RhoA. <i>T</i>: amount of Ras and RhoA in whole cell lysates. <b>D</b>. ZA decreased Ras activity, measured as Ras-GTP amount, and phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2 amount. GAPDH data are shown to confirm equivalent protein loading. <b>E</b>. MDR+ cells had significantly higher amounts of RhoA-GTP (<i>open bars</i>) and RhoA kinase (<i>hatched bars</i>) than MDR− cells (*p<0.005); ZA decreased both RhoA-GTP and RhoA kinase <i>vs</i> untreated (CTRL) cells (HT29-dx: °p<0.02; HMM:^◊p<0.02). The results shown in panels <b>C</b> and <b>D</b> are representative of 3 experiments. In panels <b>A</b>, <b>B</b>, and <b>E</b> the results represent the mean ± SD of 3 experiments.</p

ZA restores doxorubicin-induced cytotoxicity and ICD in MDR+ tumor cells.

<p>MDR− HT29 and MDR+ HT29-dx, and HMM cells were incubated for 48 h without (CTRL) or with 1 µmol/L ZA, for 24 h with 1 µmol/L Dox, for 48 h with 1 µmol/L ZA, followed by 1 µmol/L Dox for additional 24 h (ZA+Dox). <b>A</b>. LDH release. Dox alone and ZA+Dox induced a significant cytotoxicity in HT29 cells (*p<0.005). ZA+Dox induced a significant increase of cytotoxicity in HT29-dx (°p<0.05) and HMM cells (^◊p<0.02). <b>B</b>. Western blot analysis of extracellular HMGB1. Dox alone and ZA+Dox in HT29 cells, ZA+Dox in HT29-dx and HMM cells induced the release of HMGB1 in the cell culture medium. Red Ponceau staining was used to check the equal loading of proteins. <b>C</b>. Extracellular release of ATP. Dox and ZA+Dox induced a significant increase of extracellular ATP in HT29 cells (*p<0.01). ZA+Dox elicited a significant release of ATP in HT29-dx (°p<0.002) and HMM cells (^◊p<0.001). <b>D</b>. Cell surface CRT exposure. Dox and ZA+Dox induced a significant CRT exposure in HT29 cells (*p<0.001). ZA+Dox induced a significant CRT exposure in HT29-dx (°p<0.001) and HMM cells (^◊p<0.005). For panels <b>A, C</b> and <b>D</b> bars represent the mean ± SD of 3 independent experiments. For panel <b>B</b> the results are representative data from one of 2 experiments.</p

ZA-induced inhibition of HIF-1α activity and Pgp expression in MDR+ cancer cells.

<p><b>A</b>. Detection of phosphorylated (pHIF-1α) and total HIF-1α in MDR− HT29, and MDR+ HT29-dx and HMM cells after 48-hour incubation without (CTRL) or with 1 µmol/L ZA (ZA). <b>B</b>. HIF-1 activity was higher (*p<0.001) in MDR+ HT29-dx and HMM cells than HT29 cells. After ZA treatment (as reported in <b>A</b>), a significant decrease of HIF-1 activity was observed in HT29-dx (°p<0.001) and HMM cells (^◊p<0.001). <b>C</b>. Chromatin immunoprecipitation of HIF-1α on <i>mdr1</i> promoter in MDR− and MDR+ cells, treated as reported in <b>a</b>. <i>pro mdr1</i>: PCR product from immunoprecipitated samples. <i>Input</i>: PCR product from non immunoprecipitated samples (genomic DNA). <i>no Ab</i>: samples incubated in the absence of anti-HIF-1α antibody. “-”: blank. <b>D</b>. Western blotting detection of Pgp in cells treated as described in <b>A</b>. <b>E</b>. Intracellular doxorubicin was measured spectrofluorimetrically: significantly lower concentrations were detected in HT29-dx and HMM <i>vs</i> HT29 cells (*p<0.002), significantly higher concentrations in ZA-treated cells <i>vs</i> untreated (CTRL) counterparts (HT29-dx: °p<0.02; HMM: ^◊p<0.02). The results shown in panels <b>A</b>, <b>C</b> and <b>D</b> are representative of 3 experiments. For panels <b>B</b> and <b>E</b> the bars represent the mean ± SD of 3 independent experiments.</p

DataSheet_1_Immune dysfunctions affecting bone marrow Vγ9Vδ2 T cells in multiple myeloma: Role of immune checkpoints and disease status.docx

IntroductionBone marrow (BM) Vγ9Vδ2 T cells are intrinsically predisposed to sense the immune fitness of the tumor microenvironment (TME) in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS).MethodsIn this work, we have used BM Vγ9Vδ2 T cells to interrogate the role of the immune checkpoint/immune checkpoint-ligand (ICP/ICP-L) network in the immune suppressive TME of MM patients.ResultsPD-1+ BM MM Vγ9Vδ2 T cells combine phenotypic, functional, and TCR-associated alterations consistent with chronic exhaustion and immune senescence. When challenged by zoledronic acid (ZA) as a surrogate assay to interrogate the reactivity to their natural ligands, BM MM Vγ9Vδ2 T cells further up-regulate PD-1 and TIM-3 and worsen TCR-associated alterations. BM MM Vγ9Vδ2 T cells up-regulate TIM-3 after stimulation with ZA in combination with αPD-1, whereas PD-1 is not up-regulated after ZA stimulation with αTIM-3, indicating a hierarchical regulation of inducible ICP expression. Dual αPD-1/αTIM-3 blockade improves the immune functions of BM Vγ9Vδ2 T cells in MM at diagnosis (MM-dia), whereas single PD-1 blockade is sufficient to rescue BM Vγ9Vδ2 T cells in MM in remission (MM-rem). By contrast, ZA stimulation induces LAG-3 up-regulation in BM Vγ9Vδ2 T cells from MM in relapse (MM-rel) and dual PD-1/LAG-3 blockade is the most effective combination in this setting.DiscussionThese data indicate that: 1) inappropriate immune interventions can exacerbate Vγ9Vδ2 T-cell dysfunction 2) ICP blockade should be tailored to the disease status to get the most of its beneficial effect.</p