Vinorelbine, cyclophosphamide and 5-FU effects on the circulating and intratumoural landscape of immune cells improve anti-PD-L1 efficacy in preclinical models of breast cancer and lymphoma.

BACKGROUND: Anti-PD-1 and anti-PD-L1 checkpoint inhibitors (CIs) are clinically active in many types of cancer. However, only a minority of patients achieve a complete and/or long-lasting clinical response. We studied the effects of different doses of three widely used, orally active chemotherapeutics (vinorelbine, cyclophosphamide and 5-FU) over local and metastatic tumour growth, and the landscape of circulating and tumour-infiltrating immune cells involved in CI activity. METHODS: Immunocompetent Balb/c mice were used to generate models of breast cancer (BC) and B-cell lymphoma. Vinorelbine, cyclophosphamide and 5-FU (alone or in combination with CIs), were given at low-dose metronomic, medium, or maximum tolerable dosages. RESULTS: Cyclophosphamide increased circulating myeloid derived suppressor cells (MDSC). Vinorelbine, cyclophosphamide and 5-FU reduced circulating APCs. Vinorelbine and cyclophosphamide (at medium/high doses) reduced circulating Tregs. Cyclophosphamide (at low doses) and 5-FU (at medium doses) slightly increased circulating Tregs. Cyclophosphamide was the most potent drug in reducing circulating CD3+CD8+ and CD3+CD4+ T cells. Vinorelbine, cyclophosphamide and 5-FU reduced the number of circulating B cells, with cyclophosphamide showing the most potent effect. Vinorelbine reduced circulating NKs, whereas cyclophosphamide and 5-FU, at low doses, increased circulating NKs. In spite of reduced circulating T, B and NK effector cells, preclinical synergy was observed between chemotherapeutics and anti-PD-L1. Most-effective combinatorial regimens where associated with neoplastic lesions enriched in B cells, and, in BC-bearing mice (but not in mice with lymphoma) also in NK cells. CONCLUSIONS: Vinorelbine, cyclophosphamide and 5-FU have significant preclinical effects on circulating and tumour-infiltrating immune cells and can be used to obtain synergy with anti-PD-L1.

Adipose Progenitor Cell Secretion of GM-CSF and MMP9 Promotes a Stromal and Immunological Microenvironment That Supports Breast Cancer Progression.

A cell population with progenitor-like phenotype (CD45-CD34+) resident in human white adipose tissue (WAT) is known to promote the progression of local and metastatic breast cancer and angiogenesis. However, the molecular mechanisms of the interaction have not been elucidated. In this study, we identified two proteins that were significantly upregulated in WAT-derived progenitors after coculture with breast cancer: granulocyte macrophage colony-stimulating factor (GM-CSF) and matrix metallopeptidase 9 (MMP9). These proteins were released by WAT progenitors in xenograft and transgenic breast cancer models. GM-CSF was identified as an upstream modulator. Breast cancer-derived GM-CSF induced GM-CSF and MMP9 release from WAT progenitors, and GM-CSF knockdown in breast cancer cells neutralized the protumorigenic activity of WAT progenitors in preclinical models. GM-CSF neutralization in diet-induced obese mice significantly reduced immunosuppression, intratumor vascularization, and local and metastatic breast cancer progression. Similarly, MMP9 inhibition reduced neoplastic angiogenesis and significantly decreased local and metastatic tumor growth. Combined GM-CSF neutralization and MMP9 inhibition synergistically reduced angiogenesis and tumor progression. High-dose metformin inhibited GM-CSF and MMP9 release from WAT progenitors in in vitro and xenograft models. In obese syngeneic mice, metformin treatment mimicked the effects observed with GM-CSF neutralization and MMP9 inhibition, suggesting these proteins as new targets for metformin. These findings support the hypothesis that GM-CSF and MMP9 promote the protumorigenic effect of WAT progenitors on local and metastatic breast cancer. Cancer Res; 77(18); 5169-82. ©2017 AACR.

Aspirin and atenolol enhance metformin activity against breast cancer by targeting both neoplastic and microenvironment cells.

Metformin can induce breast cancer (BC) cell apoptosis and reduce BC local and metastatic growth in preclinical models. Since Metformin is frequently used along with Aspirin or beta-blockers, we investigated the effect of Metformin, Aspirin and the beta-blocker Atenolol in several BC models. In vitro, Aspirin synergized with Metformin in inducing apoptosis of triple negative and endocrine-sensitive BC cells, and in activating AMPK in BC and in white adipose tissue (WAT) progenitors known to cooperate to BC progression. Both Aspirin and Atenolol added to the inhibitory effect of Metformin against complex I of the respiratory chain. In both immune-deficient and immune-competent preclinical models, Atenolol increased Metformin activity against angiogenesis, local and metastatic growth of HER2+ and triple negative BC. Aspirin increased the activity of Metformin only in immune-competent HER2+ BC models. Both Aspirin and Atenolol, when added to Metformin, significantly reduced the endothelial cell component of tumor vessels, whereas pericytes were reduced by the addition of Atenolol but not by the addition of Aspirin. Our data indicate that the addition of Aspirin or of Atenolol to Metformin might be beneficial for BC control, and that this activity is likely due to effects on both BC and microenvironment cells.

The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells.

The human white adipose tissue (WAT) contains progenitors with cooperative roles in breast cancer (BC) angiogenesis, local and metastatic progression. The biguanide Metformin (Met), commonly used for Type 2 diabetes, might have activity against BC and was found to inhibit angiogenesis in vivo. We studied Met and another biguanide, phenformin (Phe), in vitro and in vivo in BC models. In vitro, biguanides activated AMPK, inhibited Complex 1 of the respiratory chain and induced apoptosis of BC and WAT endothelial cells. In coculture, biguanides inhibited the production of several angiogenic proteins. In vivo, biguanides inhibited local and metastatic growth of triple negative and HER2+ BC in immune-competent and immune-deficient mice orthotopically injected with BC. Biguanides inhibited local and metastatic BC growth in a genetically engineered murine model model of HER2+ BC. In vivo, biguanides increased pimonidazole binding (but not HIF-1 expression) of WAT progenitors, reduced tumor microvessel density and altered the vascular pericyte/endothelial cell ratio, so that cancer vessels displayed a dysplastic phenotype. Phe was significantly more active than Met both in vitro and in vivo. Considering their safety profile, biguanides deserve to be further investigated for BC prevention in high-risk subjects, in combination with chemo and/or targeted therapy and/or as post-therapy consolidation or maintenance therapy for the prevention of BC recurrence.

A subpopulation of circulating endothelial cells express CD109 and is enriched in the blood of cancer patients.

BACKGROUND: The endothelium is not a homogeneous organ. Endothelial cell heterogeneity has been described at the level of cell morphology, function, gene expression, and antigen composition. As a consequence of the genetic, transcriptome and surrounding environment diversity, endothelial cells from different vascular beds have differentiated functions and phenotype. Detection of circulating endothelial cells (CECs) by flow cytometry is an approach widely used in cancer patients, and their number, viability and kinetic is a promising tool to stratify patient receiving anti-angiogenic treatment. METHODOLOGY/PRINCIPAL FINDINGS: Currently CECs are identified as positive for a nuclear binding antigen (DNA+), negative for the pan leukocyte marker CD45, and positive for CD31 and CD146. Following an approach recently validated in our laboratory, we investigated the expression of CD109 on CECs from the peripheral blood of healthy subject and cancer patients. The endothelial nature of these cells was validated by RT-PCR for the presence of m-RNA level of CDH5 (Ve-Cadherin) and CLDN5 (Claudin5), two endothelial specific transcripts. Before treatment, significantly higher levels of CD109+ CECs and viable CD109+CECs were found in breast cancer patients and glioblastoma patients compared to healthy controls, and their number significantly decreased after treatment. Higher levels of endothelial specific transcripts expressed in developing endothelial cells CLEC14a, TMEM204, ARHGEF15, GPR116, were observed in sorted CD109+CECs when compared to sorted CD146+CECs, suggesting that these genes can play an important role not only during embryogenesis but also in adult angiogenesis. Interestingly, mRNA levels of TEM8 (identified as Antrax Toxin Receptor1, Antrax1) were expressed in CD109+CECs+ but not in CD146+CECs. CONCLUSION: Taken together our results suggest that CD109 represent a rare population of circulating tumor endothelial cells, that play a potentially useful prognostic role in patients with glioblastoma. The role of CD109 expression in cancer vessel-specific endothelial cells deserves to be further investigated by gene expression studies.