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Background. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a well-established diagnostic procedure for evaluating hilar and mediastinal lymphadenopathies and is the gold standard for lung cancer diagnosis and staging. Recent studies assessed the effectiveness of the 19-G flex needle in obtaining larger EBUS-TBNA samples, and prospective small series gave similar results in terms of diagnostic yield when testing different gauge needles. The lack of homogeneity between series and the small sample size of some prospective cohorts poses a limit to the validity of those results. This prospective controlled study compared the 19-G flex and 22-G needles in terms of diagnostic yield. An objective laboratory method was used to count cells and compare the two needles’ cytologic yields. Material. A prospective controlled study was conducted on 90 patients undergoing EBUS-TBNA for the diagnosis of hilar and mediastinal lymphadenopathies. The institutional ethic committee (IEO573) approved the study, and informed consent was obtained from all patients. Results. A total of 90 patients were enrolled in this study, 84.4% of whom were diagnosed with malignancy and 15.6% with non-neoplastic disease. Sensitivity for malignancy was 93.4% (CI: 87.4–97.1%) for the 19-G needle and 92.6% (CI: 86.3–96.5%) for the 22-G needle (p = 0.80). The percentage of malignant cells in the cell block was 63.9% and 61.5% for the 22-G and 19-G needles, respectively. The cell count assessed by flow cytometry was 2071 cells/µL (IQR: 600,2265) with the 22-G needle and 2761 cells/µL (IQR: 505,3250) with the 19-G needle (p = 0.79). The malignant cell count was 0.05 × 103 cells/µL with the 22-G and 0.08 × 103 cells/µL with the 19-G needle (p = 0.70). There was no difference in the presence of tissue cores in the samples, and rapid on-site evaluation (ROSE) cellularity was comparable between the two needles. Conclusions. The 19-G flex EBUS-TBNA needle is comparable to the 22-G needle in terms of diagnostic yield for cyto-histological evaluation of hilar and mediastinal lymphadenopathies. There is no difference between the 19-G and 22-G needle cell counts evaluated by flow cytometry.

Additional results from obese syngeneic models

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Supplementary information on metformin experiments

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Up-regulation of murine GM-CSF and MMP9 in vivo in WAT progenitors from MMTV-PyMT mice

Checkpoint inhibitors (CI) instigate anticancer immunity in many neoplastic diseases, albeit only in a fraction of patients. The clinical success of cyclophosphamide (C)-based haploidentical stem-cell transplants indicates that this drug may re-orchestrate the immune system. Using models of triple-negative breast cancer (TNBC) with different intratumoral immune contexture, we demonstrate that a combinatorial therapy of intermittent C, CI, and vinorelbine activates antigen-presenting cells (APC), and abrogates local and metastatic tumor growth by a T-cell–related effect. Single-cell transcriptome analysis of >50,000 intratumoral immune cells after therapy treatment showed a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoral Tcf1+ stem-like CD8+ T cells and altered the balance between terminally and progenitor-exhausted T cells favoring the latter. Overall, our data support the clinical investigation of this therapy in TNBC.Significance:A combinatorial therapy in mouse models of breast cancer increases checkpoint inhibition by activating antigen-presenting cells, enhancing intratumoral Tcf1+ stem-like CD8+ T cells, and increasing progenitor exhausted CD8+ T cells.

Additional results from xenograft TNBC models.

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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.

Supplementary Figures

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Additional experiments supporting cell co-culturing findings

Obesity is associated with an increased frequency, morbidity, and mortality of several types of neoplastic diseases, including postmenopausal breast cancer. We found that human adipose tissue contains two populations of progenitors with cooperative roles in breast cancer. CD45−CD34+CD31+CD13−CCRL2+ endothelial cells can generate mature endothelial cells and capillaries. Their cancer-promoting effect in the breast was limited in the absence of CD45−CD34+CD31−CD13+CD140b+ mesenchymal progenitors/adipose stromal cells (ASC), which generated pericytes and were more efficient than endothelial cells in promoting local tumor growth. Both endothelial cells and ASCs induced epithelial-to-mesenchymal transition (EMT) gene expression in luminal breast cancer cells. Endothelial cells (but not ASCs) migrated to lymph nodes and to contralateral nascent breast cancer lesions where they generated new vessels. In vitro and in vivo, endothelial cells were more efficient than ASCs in promoting tumor migration and in inducing metastases. Granulocyte colony-stimulating factor (G-CSF) effectively mobilized endothelial cells (but not ASCs), and the addition of chemotherapy and/or of CXCR4 inhibitors did not increase endothelial cell or ASC blood mobilization. Our findings suggest that adipose tissue progenitor cells cooperate in driving progression and metastatic spread of breast cancer. Cancer Res; 73(19); 5880–91. ©2013 AACR.

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Supplementary Methods, Figures 1-6, Tables A-C from Targeting Activin Receptor-Like Kinase 1 Inhibits Angiogenesis and Tumorigenesis through a Mechanism of Action Complementary to Anti-VEGF Therapies

PDF file, 105K, Supplementary Table 1, Monoclonal antibodies used in the study. Supplementary Table 2, EMT gene expression in luminal breast cancer cells ZR75-1 in transwell culture with human WAT totatl CD34+ cells, ECs and ASCs.

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Previous studies have suggested a “catalytic role” in neoplastic angiogenesis and cancer progression for bone marrow–derived endothelial progenitor cells (EPC). However, preclinical and clinical studies have shown that the quantitative role of marrow-derived EPCs in cancer vascularization is extremely variable. We have found that human and murine white adipose tissue (WAT) is a very rich reservoir of CD45-CD34+ EPCs with endothelial differentiation potential, containing a mean of 263 times more CD45-CD34+ cells/mL than bone marrow. Compared with marrow-derived CD34+ cells mobilized in blood by granulocyte colony–stimulating factor, purified WAT-CD34+ cells expressed similar levels of stemness-related genes, significantly increased levels of angiogenesis-related genes, and increased levels of FAP-α, a crucial suppressor of antitumor immunity. In vitro, WAT-CD34+ cells generated mature endothelial cells and capillary tubes as efficiently as mature mesenchymal cells. The coinjection of human WAT-CD34+ cells from lipotransfer procedures contributed to tumor vascularization and significantly increased tumor growth and metastases in several orthotopic models of human breast cancer in immunodeficient mice. Endothelial cells derived from human WAT-CD34+ cells lined the lumen of cancer vessels. These data indicate that CD34+ WAT cells can promote cancer progression and metastases. Our results highlight the importance of gaining a better understanding of the role of different WAT-derived cells used in lipotransfer for breast reconstruction in patients with breast cancer. Cancer Res; 72(1); 325–34. ©2011 AACR.

PDF file, 1926K, Supplementary Fig. 1 Decreasing levels of CD34 expression among some ASCs. Supplementary Fig. 2: HEV-specific MECA 79 expression. Supplementary Fig. 3: Renal capsule invasion assay. Supplementary Fig. 4: z-stack 3D studies. Supplementary Fig. 5: z-stack 3D studies (cont). Supplementary Fig. 6: EMT morphology. Supplementary Fig. 7: Cell identification in time lapse studies. Supplementary Fig. 8: CCRL2 expression in WAT cells.

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We have previously shown in triple-negative breast cancer (TNBC) models that a triple therapy (TT) including intermittent cyclophosphamide (C), vinorelbine (V), and anti-PD-1 activates antigen-presenting cells (APC) and generates stem like-T cells able to control local and metastatic tumor progression. In the present manuscript, we report the generation of a highly aggressive, anti-PD-1 resistant model of a high-grade, Myc-driven B-cell non-Hodgkin’s lymphoma (NHL) that can be controlled in vivo by TT but not by other chemotherapeutic agents, including cytarabine (AraC), platinum (P), and doxorubicin (D). The immunological memory elicited in tumor-bearing mice by TT (but not by other treatments) can effectively control NHL re-challenge even at very high inoculum doses. TT re-shaped the landscape of circulating innate NK cells and adaptive immune cells, including B and T cells, and significantly reduced exhausted CD4+ and CD8+ TIM3+PD-1+ T cells in the spleens of treated mice.

Background: The success of targeted therapies in the treatment of pancreatic neuroendocrine tumors has emphasized the strategy of targeting angiogenesis and the PI3K/AKT/mTOR pathway. However, the major challenge in the targeted era remains the early identification of resistant tumors especially when the efficacy is rarely associated to a clear tumor shrinkage at by imaging assessment. Methods: In this prospective study (NCT02305810) we investigated the predictive and prognostic role of soluble biomarkers of angiogenesis turnover (VEGF, bFGF, VEGFR2, TSP-1) circulating endothelial cells and progenitors, in 43 patients with metastatic panNET receiving everolimus. Results: Among all tested biomarkers, we found a specific subpopulation of circulating cells, CD31+CD140b-, with a significantly increased tumor progression hazard for values less or equal to the first quartile. Conclusion: Our study suggested the evidence that circulating cells might be surrogate biomarkers of angiogenesis activity in patients treated with everolimus and their baseline levels can be correlated with survival. However, further studies are now needed to validate the role of these cells as surrogate markers for the selection of patients to be candidates for antiangiogenic treatments.

Checkpoint inhibitors (CI) instigate anticancer immunity in many neoplastic diseases, albeit only in a fraction of patients. The clinical success of cyclophosphamide (C)-based haploidentical stem-cell transplants indicates that this drug may re-orchestrate the immune system. Using models of triple-negative breast cancer (TNBC) with different intratumoral immune contexture, we demonstrate that a combinatorial therapy of intermittent C, CI, and vinorelbine activates antigen-presenting cells (APC), and abrogates local and metastatic tumor growth by a T-cell–related effect. Single-cell transcriptome analysis of >50,000 intratumoral immune cells after therapy treatment showed a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoral Tcf1+ stem-like CD8+ T cells and altered the balance between terminally and progenitor-exhausted T cells favoring the latter. Overall, our data support the clinical investigation of this therapy in TNBC. Significance: A combinatorial therapy in mouse models of breast cancer increases checkpoint inhibition by activating antigen-presenting cells, enhancing intratumoral Tcf1+ stem-like CD8+ T cells, and increasing progenitor exhausted CD8+ T cells.

Background During the course of COVID-19, the disease caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thrombotic phenomena and/or diffuse vascular damage are frequent, and viral elements have been observed within endothelial cells. Objectives CD146 + circulating endothelial cells (CD146 + CECs) and their progenitors (CEPs) are increased in cardiovascular, thrombotic, infectious, and cancer diseases. The present study was designed to investigate their kinetics in novel coronavirus (COVID-19) patients. Methods We used a validated flow cytometry procedure to enumerate viable and apoptotic CD146 + CECs and CEPs in COVID-19 patients during the course of the disease and in patients who recovered. Results Viable CEPs per milliliter were significantly increased in COVID-19 patients compared with healthy controls. This increase was observed in patients with mild symptoms and not further augmented in patients with severe symptoms. In patients who recovered, CEPs decreased, but were in a range still significantly higher than normal controls. Regarding mature CD146 + CECs, in COVID-19 patients, their absolute number was similar to those observed in healthy controls, but the viable/apoptotic CD146 + CEC ratio was significantly different. Both mild and severe COVID-19 patients had significantly less apoptotic CD146 + CECs compared with healthy controls. Patients who recovered had significantly less CD146 + CECs per milliliter when compared with controls as well as to mild and severe COVID-19 patients. A positive correlation was found between the copies of SARS-CoV-2 RNA in the cellular fraction and apoptotic CEPs per milliliter in severe COVID-19 patients. Conclusions CD146 + CECs and CEPs might be investigated as candidate biomarkers of endothelial damage in COVID-19 patients.

Breast cancer (BC) constitutes a major health problem worldwide, making it the most common malignancy in women. Current treatment options for BC depend primarily on histological type, molecular markers, clinical aggressiveness and stage of disease. Immunotherapy, such as anti-PD-1, have shown combinatorial clinical activity with chemotherapy in triple negative breast cancer (TNBC) delineating some therapeutic combinations as more effective than others. However, a clear overview of the main immune cell populations involved in these treatments has never been provided.Here, an assessment of the immune landscape in the tumour microenvironment (TME) of two TNBC mouse models (4T1 and EMT6 cell lines) has been performed using single-cell RNA sequencing (scRNA-seq) technology. Specifically, immune cells were evaluated in untreated conditions and after being treated with chemotherapy or immunotherapy used as single agents or in combination. A decrease of regulatory T cells, compared to the untreated TME, was found in treatments with in vivo efficacy as well as γδ T cells, which have a pro-tumoral activity in mice. Focusing on Cd8 T cells, across all the conditions, a general increase of exhausted-like Cd8 T cells was confirmed in pre-clinical treatments with low efficacy; on the other hand, an opposite trend was found for the proliferative Cd8 T cells. Regarding macrophages, M2-like cells were found enriched in treatments with low efficacy while opposite behaviour was associated with M1-like macrophages. For both cell lines, similar proportions of B cells were detected with an increase of proliferative B cells in treatments that involved cisplatin in combination with anti-PD-1. The fine-scale characterization of the immune TME in this work can lead to new insights on the diagnosis and treatment of TNBC for a possible application at the clinical level.

The role and regulation of innate immune cells is poorly understood in B-cell non-Hodgkin lymphoma (NHL). As natural killer (NK) cells, helper innate lymphoid cells (ILCs) are lymphocytes endowed with either anti- or pro-tumour activity and involved in inflammatory processes. In our ex vivo analysis of NK cells and ILCs from NHL patients, we observed that, in comparison to healthy donors (HD), the frequency of the cytotoxic subset of NK cells, the CD16

Checkpoint inhibitors (CIs) such as anti-PD-1 and anti PD-L1 have shown combinatorial clinical activity with chemotherapy in triple negative breast cancer (TNBC), but only in a minority of patients and only for a limited period of time. Moreover, it is currently unclear what chemotherapeutic drug is the most appropriate to combine with CIs in TNBC patients. We have investigated at the single cell level the transcriptome and the trajectories of more than 50,000 innate and adaptive intratumoral immune cells in two syngeneic, immune competent, orthotopic murine models of local and metastatic TNBC. Mice injected with 4T1 cells had a predominant lymphoid infiltrate, mice injected with EMT6 cells had a predominant myeloid infiltrate. Mice were treated with CIs and several different types of chemotherapeutics, alone or in combinations. In both models, capecitabine (alone or with CIs) was the less effective drug. Platinum, doxorubicin and taxanes showed synergy with CIs and had superimposable activity. Intermittent, medium dosage cyclophosphamide (CTX) plus vinorelbine and CIs was the most active combinatorial therapy (Falvo et al, Cancer Research 2021). Vinorelbine activated antigen presenting cells and CTX generated new T cell clones including stem cell-like TCF1+ CD8+ T cells. Treatments with most in vivo efficacy were associated to a decrease of regulatory T cells and of gamma delta T cells, which were found to have a pro-tumoral activity in these murine models, likely due to IL-17 expression in the neoplastic microenvironment. An increase of several different clusters of exhausted-like CD8+ T cells was observed in pre-clinical treatments with low efficacy; an opposite trend was found for several clusters of proliferative CD8+ T cells in treatments with high in vivo efficacy. Regarding macrophages, M2-like cells were enriched after treatments with low efficacy, while an opposite behaviour was found in M1-like macrophages. Interestingly, we observed a significant increase of an M1-like cluster with high expression of the Ly6c1/Ly6c2 gene in mice successfully treated with vinorelbine, CTX and CIs. For both cell lines the percentage of plasma B cells increased after in vivo treatments with high efficacy. In particular, the most effective treatment significantly increased the frequency of germinal B cells, which were absent in untreated tumors. These data can lead to new insights on the diagnosis and treatment of TNBC and to possible clinical applications. Citation Format: Laura Carpen, Paolo Falvo, Stefania Orecchioni, Giulia Mitola, Roman Hillje, Saveria Mazzara, Patrizia Mancuso, Stefano Pileri, Alessandro Raveane, Francesco Bertolini. A single-cell RNA atlas of innate and adaptive intratumoral immunity in triple negative breast cancer during chemo- and immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1712.

Checkpoint inhibitors (CI) instigate anticancer immunity in many neoplastic diseases, albeit only in a fraction of patients. The clinical success of cyclophosphamide (C)-based haploidentical stem-cell transplants indicates that this drug may re-orchestrate the immune system. Using models of triple-negative breast cancer (TNBC) with different intratumoral immune contexture, we demonstrate that a combinatorial therapy of intermittent C, CI, and vinorelbine activates antigen-presenting cells (APC), and abrogates local and metastatic tumor growth by a T-cell-related effect. Single-cell transcriptome analysis of50,000 intratumoral immune cells after therapy treatment showed a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoral Tcf1

Venetoclax is a potent, orally available, small molecule B-cell lymphoma 2 (Bcl-2) Homology 3 (BH3) mimetic, capable of promoting apoptosis through selective inhibition of pro‐survival protein BCL2...

During the course of Covid-19, the disease caused by the new Coronavirus SARS-CoV-2, thrombotic phenomena and/or diffuse vascular damage are frequent, and viral elements have been observed within endothelial cells. Circulating endothelial cells (CECs) and their progenitors (CEPs) are increased in cardiovascular, thrombotic, infectious and cancer diseases. Using a validated flow cytometry procedure, we found that viable CEPs/mL were significantly increased in Covid-19 patients compared to healthy controls. This increase was observed in patients with mild symptoms and not further augmented in patients with severe symptoms. In patients who recovered, CEPs decreased, but were in a range still significantly higher than normal controls. Regarding mature CECs, in Covid-19 patients their absolute number was similar to those observed in healthy controls, but the viable/apoptotic CEC ratio was significantly different. Both mild and severe Covid-19 patients had significantly more viable CECs compared to healthy controls. Patients who recovered had significantly less CECs/mL when compared to controls as well as to mild and severe Covid-19 patients. A positive correlation was found between the copies of SARS-CoV-2 RNA in the cellular fraction and apoptotic CEPs/mL in severe Covid-19 patients. These findings suggest that CECs and CEPs might be investigated as candidate biomarkers of endothelial damage in Covid-19 patients.

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.

Checkpoint inhibitors (CIs) instigate anticancer immunity, albeit only in a fraction of patients. Triple negative breast cancer (TNBC) is among the most aggressive and lethal BC types, and currently available therapies have an unsatisfactory clinical impact. The association of CIs with chemotherapy had encouraging results in randomized TNBC trials, but so far there has been no clear evidence of what should be considered the best combinatorial therapy. In B-cell non-Hodgkin's lympoma (NHL), CIs' clinical activity has been so far unsatisfactory. We designed the current study to define what could be considered the most efficient combinatorial regimen of chemotherapy plus CI in TNBC and NHL. We investigated different options including platinum (P), doxorubicin (D), taxol (T), vinorelbine (V), and cyclophosphamide ( C), i.e. the most effective chemotherapy drugs for these diseases. Capecitabine was not considered as found not to be effective in combination with CIs. As TNBC patients have a large intratumoral immune cell heterogeneity, we investigated two immune competent TNBC models: 4T1 with a predominant lymphoid intratumoral infiltrate and EMT6 with a predominant myeloid infiltrate. A C57BL/6 Eμ-myc transgenic NHL model, with a predominant lymphatic dissemination pattern, was also studied. We found that 1) Intermittent, medium-dosage C (C140), was more effective than other combinatorial regimens including the CI anti-PD1 plus low-dose C, or regular-dose T, D, or P. The association of V and anti-PD1 further increased the preclinical efficacy of C140; 2) both CD3+CD4+ and CD3+CD8+ T cells (at variance with other subsets of NK and myeloid cells) were needed to control local and metastatic neoplastic growth in mice treated with C140, V and anti-PD1. 3) The combinatorial therapy including C140, V and anti-PD1 was the most efficient in activating antigen presenting cells (APCs), and the investigation of TCR repertoire indicated that this therapy generated a significantly larger clonal expansion of both CD3+CD4+ and CD3+CD8+ T cells. 4) Single-cell transcriptome analysis of >50,000 intratumoural immune cells showed after C140, V and anti-PD1 therapy a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or an antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoural tcf1+ stem-like CD8+ T-cells and altered the balance between terminally and progenitor exhausted T-cells, favoring the latter. These data support the clinical investigation of this therapy in TNBC and NHL, and we are currently investigating the related transcriptional mechanisms inducing APC activation and T cell rewiring. Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Giulia Mitola, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, PierGiuseppe Pelicci, Francesco Bertolini. A single-cell atlas of the effect of chemotherapeutics over intratumoral immune cells reveals that combining an alkylating agent and a vinca alkaloid can activate antigen presenting cells and increase tcf1+ stem-like CD8+ T-cells, thus improving anti-PD-1 efficacy in triple negative breast cancer and lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1653.

The E3 ubiquitin ligase (E3) WWP1 is an oncogenic factor implicated in the maintenance of different types of epithelial cancers. The role of WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) in haematological neoplasms remains unknown. Acute myeloid leukaemia (AML) is characterized by the expansion of malignant myeloid cells blocked at different stages of differentiation. Here we report that the expression of WWP1 is significantly augmented in a large cohort of primary AML patients and in AML cell lines, compared with haematopoietic cells from healthy donors. We show that WWP1 inactivation severely impairs the growth of primary AML blasts and cell lines in vitro. In vivo, we observed a reduced leukaemogenic potential of WWP1-depleted AML cells upon transplantation into immunocompromised mice. Mechanistically, WWP1 inactivation induces the accumulation of its protein substrate p27Kip1, which ultimately contributes to G0/G1 cell cycle arrest of AML blasts. In addition, WWP1 depletion triggers the autophagy signalling and reduces survival of leukaemic cells. Collectively, our findings provide molecular insights into the anti-cancer potential of WWP1 inhibition, suggesting that this E3 is a promising biomarker and druggable target in AML.

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.

Immunotherapy is a new standard first-line treatment for non-small cell lung cancers (NSCLC) with high programmed cell death-ligand 1 (PD-L1) expression (≥ 50%) and second-line treatment regardless of PD-L1 status, though not all patients benefit from this approach. Much effort is ongoing to identify robust prognostic and predictive biomarkers of response to immune checkpoint inhibitors, overcoming PD-L1 that appears limited in its ability to discriminate patient candidates to this new class of anticancer agents. The purpose of this research study is to identify potential new biomarkers for immunotherapy in lung cancer.Fifty-three consecutive patients with advanced NSCLC treated with nivolumab were enrolled in the study. All the patients received a blood analysis looking for the relationship between different populations of baseline white blood cells and granulocytic myeloid-derived suppressor cells (Gr-MDSC) detected by flow cytometry, to identify and characterize patients with poor likelihood of benefit from nivolumab in NSCLC second-line setting, regardless of clinical feature and PDL1 expression.Univariate analysis showed that high baseline levels of Gr-MDSC and low baseline CD8/Gr-MDSC ratio are associated with significantly better (P = 0.02) response to immunotherapy treatment. Log-rank tests suggested a significant improvement in OS and PFS with high baseline levels of Gr-MDSC levels (≥ 6 cell/μl), low absolute neutrophil count ( 5840/μl), high eosinophil count ( 90 /μl), and NLR 3. The multivariate analysis showed a statistically significant improvement for PFS (P = 0.003) and OS (P = 0.05) in favour of the identified good prognostic Gr-MDSC-linked asset group, compared with the poor prognosis group.The role of Gr-MDSC appears interesting as a potential biomarker in NSCLC patients receiving immune-checkpoint inhibitors. Further analyses are needed to confirmed and study in deep the role of these particular cells and their role in cancer response and progression during ICI therapy.

The clinical success of cyclophosphamide (Cy)-based haploidentical stem cell transplants in hematological malignancies indicates that this drug has the potential to reorchestrate the immune system against cancer cells. Along a similar way, Cy is administered before the infusion of CAR-T cells to improve their clinical efficacy. We have previously found that daily, low/medium-dose Cy (20mg/Kg), in association with vinorelbine (V) was able to improve the preclinical efficacy of checkpoint inhibitors (CIs) anti-PD-1 and anti-PD-L1 in preclinical models of breast cancer and lymphoma (Orecchioni et al, 2018). Randomized clinical trials have recently indicated that the addition of CIs to other chemotherapy drugs such as taxanes (T), doxorubicin (D), or platinum (P) might be beneficial in triple negative breast cancer (TNBC) patients. Thus, we designed the present study in two orthotopic, immunocompetent, local and metastatic models of TNBC (CI-resistant 4T1 and EMT6, moderately responsive to CIs) to investigate whether Cy and V, in different schedules and doses, were more effective than T, D, or P. In vivo studies in local and metastatic models using both 4T1 and EMT6 TNBC cells clearly indicated that intermittent (ie every 6 days), medium-dosage (140 mg/Kg) Cy, was more effective than any other combinatorial regimens including CIs plus daily low/medium-dose Cy, or T, D, or P at their regular dosages. The association of V further increased the preclinical efficacy of intermittent Cy plus CIs, and abrogated tumor growth in both 4T1 and EMT6 models. In vivo studies with neutralizing monoclonal antibodies targeting in separate experiments T, B, NK, and myeloid cells demonstrated that CD3+CD4+, CD3+CD8+ T cells as well as CD11b+ monocyte/macrophage dendritic cells were crucial to abrogate tumor growth in TNBC-bearing mice treated with intermittent Cy, V and CIs. Single-cell transcriptome analysis of more than 60,000 intratumoral immune cells and flow cytometry studies in circulating and intratumoral subsets of immune cells indicated that V promoted the generation and maturation of myeloid APC cells, and that intermittent Cy generated new clones of tumor-infiltrating CD3+CD4+ and CD3+CD8+ TCR alpha beta cells. After treatment with intermittent Cy, V and CIs, intratumoral immune cells showed a unique gene signature of 42 genes (CD3D, Ptprc, CD69, Lat, Lck, CD2, CD28, CD27, and CD3E among others) suggestive of a change in morphology and behavior resulting from exposure to a mitogen, cytokine, chemokine, cellular ligand, or an antigen for which it is specific, as well as APC-to-T cell adhesion. Taken together, our data support the hypothesis that APC priming and T cell clonal replacement can significantly improve CI efficacy in vivo in TNBC models. We are further investigating the Cy-related mechanisms inducing intratumoral T-cell clonal replacement and how these T cells cooperate with V-induced APCs. Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, Francesco Bertolini. Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 900.

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.

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Checkpoint inhibitors (CIs) have shown an unprecedented clinical activity in a large variety of cancer types, but only in a fraction of patients. To increase the number of responders, preclinical models are studied to define effective combinatorial regimens and the best window of therapeutic opportunities. We recently described a synergy between anti-PD-1 and -PD-L1 CIs and several types and dosages of chemotherapy in immunocompetent models of triple negative breast cancer (TNBC), likely due to unique effects of chemotherapeutics over circulating and tumor-infiltrating, suppressive, antigen-presenting and effector immune cell populations (Orecchioni et al, Br J Cancer 2018). To find a high-order drug combination, we have further studied by multiparametric flow cytometry and single-cell transcriptomic a panel of different chemotherapy drugs (including microtubular poisons, alkylating agents, topoisomerase inhibitors and antimetabolites) used in vivo at different dosages and schedule. Our aim was to define a combination, dose and schedule able to promote a) dendritic cell maturation, b) release and c) uptake of cancer-associated antigens from targeted neoplastic cells, d) cross-priming between antigen-presenting cells (APCs) and T cells, e) NK cell activation, and f) inhibition of suppressor immune cell populations. Vinca alkaloids vinblastine and vinorelbine (V), at low-medium dosages, were the most effective in a), b), c) and d). After V administration, Tregs were reduced, and circulating and tumor-infiltrating APCs showed a “maturation to activation” program with increased expression of several antigens including CD40, CD80, and CD86. The alkylating agent cyclophosphamide (CTX) targeted Tregs, mobilized APC progenitors, activated and increased the number of circulating and intratumoral NK cells. These effects were significantly increased when CTX was administered in an intermittent fashion (every 6 days) at low-medium doses. As the APC-NK cell axis has been recently found to be pivotal in CI-mediated, anti-tumor cell immunity, our data suggested a preclinical trial in immunocompetent orthotopic models of metastatic (post-mastectomy) BALB mice injected with 4T1 or EMT-6 TNBC cells. Among a large panel of different combinations and dosages, the sequential administration of V, intermittent CTX and anti-PD-L1 was the only able to completely abrogate TNBC local and metastatic growth, and this effect was not observed in T and NK cell-depleted mice. Taken together with recent data from randomized, combinatorial studies in TNBC patients (Schmid et al, NEJM 2018), our findings suggest that CIs might be included in future combinatorial regimens aimed at improving the percentage of patients receiving a clinical benefit and prolonging the duration of this benefit. Citation Format: Stefania Orecchioni, Loredana Vecchi, Paolo Falvo, Lucilla Luzi, Patrizia Mancuso, Chiara Camisaschi, Francesco Bertolini. The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3948.