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      Radiogenomics Monitoring in Breast Cancer Identifies Metabolism and Immune Checkpoints as Early Actionable Mechanisms of Resistance to Anti-angiogenic Treatment

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          Abstract

          Anti-VEGF antibody bevacizumab has prolonged progression-free survival in several cancer types, however acquired resistance is common. Adaption has been observed pre-clinically, but no human study has shown timing and genes involved, enabling formulation of new clinical paradigms.

          In a window-of-opportunity study in 35 ductal breast cancer patients for 2 weeks prior to neoadjuvant chemotherapy, we monitored bevacizumab response by Dynamic Contrast-Enhanced Magnetic Resonance [DCE-MRI], transcriptomic and pathology.

          Initial treatment response showed significant overall decrease in DCE-MRI median K trans, angiogenic factors such ESM1 and FLT1, and proliferation. However, it also revealed great heterogeneity, spanning from downregulation of blood vessel density and central necrosis to continued growth with new vasculature. Crucially, significantly upregulated pathways leading to resistance included glycolysis and pH adaptation, PI3K-Akt and immune checkpoint signaling, for which inhibitors exist, making a strong case to investigate such combinations.

          These findings support that anti-angiogenesis trials should incorporate initial enrichment of patients with high K trans, and a range of targeted therapeutic options to meet potential early resistance pathways. Multi-arm adaptive trials are ongoing using molecular markers for targeted agents, but our results suggest this needs to be further modified by much earlier adaptation when using drugs affecting the tumor microenvironment.

          Highlights

          • DCE-MRI two weeks post-treatment of primary breast cancer shows heterogeneity of initial response to bevacizumab.

          • Gene expression shows heterogeneous changes but an overall decrease in angiogenic factors ESM1 and FLT1, and proliferation.

          • Increased PI3K-Akt and immune checkpoint signaling suggests combination of Bevacizumab with inhibitors of these pathways.

          • Increased PI3K-Akt and immune checkpoint signaling suggests combination of Bevacizumab with inhibitors of these pathways.

          Available evidence is that the anti-angiogenic treatment Bevacizumab currently has a limited range of indications in breast cancer compared to other cancers. Here, we show that multimodality dynamic monitoring of treatment response allow classification of patient's response as early as two weeks after treatment, and reveals new mechanisms associated with resistance. The implications, are that the role of bevacizumab in neoadjuvant breast cancer needs to be re-evaluated using early classification of resistant patients, but more generally that trials need to be designed prospectively, taking into account dynamic monitoring to be able to rapidly adapt available new agents for combination.

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          PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation

          Hypoxia is a common feature of solid tumors (Semenza, 2011). Hypoxic zones in tumors attract immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs; Corzo et al., 2010), tumor-associated macrophages (TAMs; Doedens et al., 2010; Imtiyaz et al., 2010), and regulatory T cells (T reg cells; Clambey et al., 2012). MDSCs are a heterogeneous group of relatively immature myeloid cells and several studies have described mechanisms of MDSC-mediated immune suppression (Gabrilovich et al., 2012). A large body of preclinical and clinical data indicates that antibody blockade of immune checkpoints can significantly enhance antitumor immunity (Pardoll, 2012; West et al., 2013). Recently, antibody-mediated blockade of preprogrammed death 1 (PD-1; Topalian et al., 2012) and its ligand, PD-L1 (Brahmer et al., 2012), was shown to result in durable tumor regression and prolonged stabilization of disease in patients with advanced cancers. PD-1, a cell surface glycoprotein with a structure similar to cytotoxic T lymphocyte antigen 4 (CTLA-4), belongs to the B7 family of co-stimulatory/co-inhibitory molecules and plays a key part in immune regulation (Greenwald et al., 2005). PD-1 has two known ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC). Although hypoxia has been shown to regulate the function and differentiation of MDSCs (Corzo et al., 2010), several major questions remain unresolved. The influence of hypoxia on the regulation of immune checkpoint receptors (PD-1 and CTLA-4) and their respective ligands (PD-L1, PD-L2, CD80, and CD86) on MDSCs remains largely obscure. Furthermore, the potential contribution of these immune checkpoint receptors and their respective ligands on MDSC function under hypoxia is still unknown. In the present study, we showed that hypoxia via hypoxia-inducible factor-1α (HIF-1α) selectively up-regulated PD-L1 on MDSCs, but not other B7 family members, by binding directly to the HRE in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia abrogated MDSC-mediated T cell suppression by modulating MDSCs cytokine production. RESULTS AND DISCUSSION Differential expression of PD-L1 on tumor-infiltrating MDSCs versus splenic MDSCs and selective up-regulation of PD-L1 in splenic MDSCs under hypoxic stress We first compared the level of expression of PD-L1 and PD-L2 between splenic MDSCs and tumor-infiltrating MDSCs from tumor-bearing mice. We found that the percentage of PD-L1+ cells was significantly higher on tumor-infiltrating MDSCs as compared with splenic MDSC in B16-F10, LLC (Fig. 1 A), CT26, and 4T1 (Fig. 1 B) tumor models. No significant difference was found in the percentage of PD-L2+ cells in splenic MDSCs as compared with tumor-infiltrating MDSCs in four tumor models tested (Fig. 1 C). We did not observe any significant difference in the expression levels of other members of the B7 family such as CD80, CD86, PD-1, and CTLA-4 on MDSCs from spleen and tumor (unpublished data). Youn et al. (2008) previously observed no significant differences in the percentage of PD-L1+ or CD80+ cells within the splenic MDSCs from tumor-bearing mice and immature myeloid cells from naive tumor-free mice. However, by comparing the expression of immune checkpoint inhibitors between splenic and tumor-infiltrating MDSCs, we showed that there is a differential expression of PD-L1 on tumor-infiltrating MDSCs. Figure 1. Tumor-infiltrating MDSCs differentially express PD-L1 as compared with splenic MDSCs, and hypoxia selectively up-regulates PD-L1 on splenic MDSCs in tumor-bearing mice. Surface expression level of PD-L1 and PD-L2 on Gr1+ CD11b+ cells (MDSCs) from (B16-F10 and LLC; A; CT26 and 4T1; B) in spleens (black dotted line histogram) and tumor (black line histogram) as compared with isotype control (gray-shaded histogram) was analyzed by flow cytometry. (C) Statistically significant differences (indicated by asterisks) between tumor-infiltrating MDSCs and splenic MDSCs are shown (*, P 20 fold for HRE-4), comparable to their binding to an established HRE in VEGF, LDHA, and Glut1 genes. To determine whether this HIF-1α site (HRE-4) was a transcriptionally active HRE, MSC-1 cells were co-transfected with pGL4-hRluc/SV40 vector and pGL3 EV, pGL3 HRE-4, or pGL3 HRE-4 MUT vectors (Fig. 3 M) and grown under normoxia or hypoxia. After 48 h, firefly and renilla luciferase activities were measured. As shown in Fig. 3 N, hypoxia significantly increased the luciferase activity of HRE-4 reporter by more than threefold as compared with normoxia. More interestingly, the luciferase activity of HRE-4 MUT was significantly decreased (>50%) as compared with HRE-4 under hypoxia (Fig. 3 N). The results presented in Figs. 3 (H–N) demonstrate that PD-L1 is a direct HIF-1α target gene in MSC-1 cells. Thus, we provide evidence here that HIF-1α is a major regulator of PD-L1 mRNA and protein expression, and that HIF-1α regulates the expression of PD-L1 by binding directly to the HRE-4 in the PD-L1 proximal promoter. Blocking PD-L1 decreases MDSC-mediated T cell suppression under hypoxia by down-regulating MDSC IL-6 and IL-10 To directly test the functional consequences of hypoxia-induced up-regulation of PD-L1 in MDSC-mediated T cell suppression, the expression of PD-L1 was blocked on ex vivo MDSCs by using anti–PD-L1 monoclonal antibody. Hypoxia increased the ability of MDSCs to suppress both specific and nonspecific stimuli-mediated T cell proliferation (Fig. 4, A and B). Interestingly, blockade of PD-L1 under hypoxia significantly abrogated the suppressive activity of MDSCs in response to both nonspecific stimuli (anti-CD3/CD28 antibody; Fig. 4 A) and specific stimuli (TRP-2(180–88) peptide; Fig. 4 B). Under hypoxia, MDSCs acquired the ability to inhibit T cell function (Fig. 4, C and D) by decreasing the percentage of IFN-γ+ CD8+ and CD4+ T cells; whereas the percentage of IFN-γ+ CD8+ (Fig. 4 C) and IFN-γ+ CD4+ T cells (Fig. 4 D) significantly increased after PD-L1 blockade under hypoxic conditions. Thus, the immune suppressive function of MDSCs enhanced under hypoxia was abrogated after blocking PD-L1, and hypoxic up-regulation of PD-L1 on MDSCs is involved in mediating the suppressive action of MDSCs, at least in part, as we were not able to completely restore T cell proliferation and function after PD-L1 blockade on MDSCs under hypoxia. Figure 4. Blockade of PD-L1 under hypoxia down-regulates MDSC IL-6 and IL-10 and enhances T cell proliferation and function. MDSCs isolated from spleens of B16-F10 tumor-bearing mice were pretreated for 30 min on ice with 5 µg/ml control antibody (IgG) or antibody against PD-L1 (PDL1 Block) and co-cultured with splenocytes under normoxia and hypoxia for 72 h. (A and B) Effect of MDSC on proliferation of splenocytes stimulated with (A) anti-CD3/CD28 coated beads or (B) TRP-2(180–88) peptide under the indicated conditions. Cell proliferation was measured in triplicates by [3H]thymidine incorporation and expressed as counts per minute (CPM). (C and D) MDSCs were cultured with splenocytes from B16-F10 mice stimulated with anti-CD3/CD28. Intracellular IFN-γ production was evaluated by flow cytometry by gating on (C) CD3+CD8+ IFN-γ+ and (D) CD3+CD4+ IFN-γ+ populations. Statistically significant differences (indicated by asterisks) are shown (**, P 95% as evaluated by FACS analysis. MDSC functional assays. For evaluation of T cell proliferation, splenocytes from B16-F10 mice were plated into U-bottom 96-well plates along with MDSCs at different ratios (50,000 MDSC:200,000 splenocytes/well). Plates were stimulated with either anti-CD3/CD28 beads (Miltenyi Biotec) or TRP-2 180–88 peptide for 72 h at 37°C. Co-cultures were pulsed with thymidine (1 µCi/well; Promega) for 16–18 h before harvesting, and [3H]thymidine uptake was counted using Packard’s TopCount NXT liquid scintillation counter and expressed as counts per minute (CPM). For assessment of T cell functions, MDSCs co-cultured with splenocytes from B16-F10 mice were stimulated with anti-CD3/CD28 beads. After 72 h, intracellular IFN-γ production was evaluated by flow cytometry by gating on CD3+CD8+ IFN-γ+ and CD3+CD4+ IFN-γ+ populations. MDSCs cytokine production (ELISA). MDSCs isolated from spleens of B16-F10 tumor-bearing mice were pretreated for 30 min on ice with 5 μg/ml control antibody (IgG) or Anti-Mouse PD-L1 (B7-H1) Functional Grade Purified antibody 5 µg/ml (clone MIH5; eBioscience; PDL1 Block) and cultured under normoxia and hypoxia for 72 h. Supernatants were collected and the secretion of IL-6, IL-10, and IL-12p70 (eBioscience) was determined by ELISA. ChIP assay. ChIP was performed with lysates prepared from MSC-1 by using SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology). SYBR Green RT-qPCR was performed using the primers detailed in Table S1. Arginase enzymatic activity and NO (nitric oxide) production. Arginase activity was measured in MDSC cell lysates, and for NO production, culture supernatants were mixed with Greiss reagent and nitrite concentrations were determined as described earlier (Youn et al., 2008). Luciferase reporter assay. A 653-bp section corresponding to mouse PD-L1 promoter containing HRE4 sequence was inserted into the NheI–XhoI sites of pGL3-Basic vector (Promega). Mutation of HRE4 was performed by site-directed mutagenesis and verified by sequencing. A 56-bp mouse PD-L1 gene sequence was inserted into the Bgl II site of pGL3-Promoter (Promega). MSC-1 cells were co-transfected with 0.2 µg of pGL4-hRluc/SV40 vector (which contains renilla luciferase sequences downstream of the SV40 promoter) and 1 µg of pGL3 empty vector, pGL3 HRE-4, or pGL3 HRE-4 MUT vectors in 6-well plates with Lipofectamine 2000 (Invitrogen) in OPTIMEM (Invitrogen) medium and grown under normoxia or hypoxia. After 48 h, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter assay (Promega) and the ratio of firefly/Renilla luciferase was determined. Statistics. Data were analyzed with GraphPad Prism. Student’s t test was used for single comparisons. Online supplemental material. Table S1 shows genomic oligonucleotide primers used for amplification of immunoprecipitated DNA samples from ChIP assays. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20131916/DC1. Supplementary Material Supplemental Material
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            Biological processes associated with breast cancer clinical outcome depend on the molecular subtypes.

            Recently, several prognostic gene expression signatures have been identified; however, their performance has never been evaluated according to the previously described molecular subtypes based on the estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2), and their biological meaning has remained unclear. Here we aimed to perform a comprehensive meta-analysis integrating both clinicopathologic and gene expression data, focusing on the main molecular subtypes. We developed gene expression modules related to key biological processes in breast cancer such as tumor invasion, immune response, angiogenesis, apoptosis, proliferation, and ER and HER2 signaling, and then analyzed these modules together with clinical variables and several prognostic signatures on publicly available microarray studies (>2,100 patients). Multivariate analysis showed that in the ER+/HER2- subgroup, only the proliferation module and the histologic grade were significantly associated with clinical outcome. In the ER-/HER2- subgroup, only the immune response module was associated with prognosis, whereas in the HER2+ tumors, the tumor invasion and immune response modules displayed significant association with survival. Proliferation was identified as the most important component of several prognostic signatures, and their performance was limited to the ER+/HER2- subgroup. Although proliferation is the strongest parameter predicting clinical outcome in the ER+/HER2- subtype and the common denominator of most prognostic gene signatures, immune response and tumor invasion seem to be the main molecular processes associated with prognosis in the ER-/HER2- and HER2+ subgroups, respectively. These findings may help to define new clinicogenomic models and to identify new therapeutic strategies in the specific molecular subgroups.
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              CTLA-4 is a second receptor for the B cell activation antigen B7

              Functional interactions between T and B lymphocytes are necessary for optimal activation of an immune response. Recently, the T lymphocyte receptor CD28 was shown to bind the B7 counter-receptor on activated B lymphocytes, and subsequently to costimulate interleukin 2 production and T cell proliferation. CTLA-4 is a predicted membrane receptor from cytotoxic T cells that is homologous to CD28 and whose gene maps to the same chromosomal band as the gene for CD28. It is not known, however, if CD28 and CTLA-4 also share functional properties. To investigate functional properties of CTLA-4, we have produced a soluble genetic fusion between the extracellular domain of CTLA-4 and an immunoglobulin C gamma chain. Here, we show that the fusion protein encoded by this construct, CTLA4Ig, bound specifically to B7-transfected Chinese hamster ovary cells and to lymphoblastoid cells. CTLA4Ig also immunoprecipitated B7 from cell surface 125I-labeled extracts of these cells. The avidity of 125I-labeled B7Ig fusion protein for immobilized CTLA4Ig was estimated (Kd approximately 12 nM). Finally, we show that CTLA4Ig was a potent inhibitor of in vitro immune responses dependent upon cellular interactions between T and B lymphocytes. These findings provide direct evidence that, like its structural homologue CD28, CTLA- 4 is able to bind the B7 counter-receptor on activated B cells. Lymphocyte interactions involving the B7 counter-receptor are functionally important for alloantigen responses in vitro.
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                Author and article information

                Contributors
                Journal
                EBioMedicine
                EBioMedicine
                EBioMedicine
                Elsevier
                2352-3964
                16 July 2016
                August 2016
                16 July 2016
                : 10
                : 109-116
                Affiliations
                [a ]Department of Oncology, University of Oxford, Oxford, UK
                [b ]Department of Engineering, University of Oxford, Oxford, UK
                [c ]Paul Strickland Scanner Centre, Northwood, Middlesex, UK
                [d ]Mount Vernon Cancer Centre, Northwood, Middlesex, UK
                Author notes
                [1]

                Contributed equally.

                [2]

                Contributed equally.

                Article
                S2352-3964(16)30322-X
                10.1016/j.ebiom.2016.07.017
                5006694
                27474395
                63628477-0156-4577-94e4-9e22432005a6
                © 2016 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 30 March 2016
                : 7 July 2016
                : 14 July 2016
                Categories
                Research Paper

                anti-angiogenic treatment,breast cancer,resistance,dce-mri,radiogenomics

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