Immunobiology of neutrophils in human colorectal cancer

Colorectal cancer (CRC) represents a major cause of cancer related death in different geographic areas. Tumor infiltration by a variety of immune cell types has consistently been observed to be associated with favorable prognosis (1). In particular, CRC infiltration by CD8+ T cells expressing memory and activation markers has repeatedly been reported to represent a favorable prognostic marker, although underlying molecular mechanisms and the antigen specificity of these cells are largely unclear. In sharp contrast, tumor infiltration by myeloid cells has classically been associated with poor prognosis in a variety of cancers of different histological origin (2). However, recent reports underline that granulocytes might participate to anti-tumor immune responses in defined cancer types (3-5). In previous studies, our group has observed that CRC infiltration by myeloid cells expressing CD16 and myeloperoxidase (MPO) is also associated with good prognosis (6;7). Based on this background, I have addressed the role of granulocyte infiltration in CRC immunobiology. By using a clinically annotated tissue microarray (TMA) including over 650 individual CRC, I have explored the prognostic significance of tumor infiltration by CD66b+ granulocytes. I found that CRC infiltration by CD66b+ granulocytes significantly correlates with favorable prognosis. Most importantly however, dense granulocyte infiltration significantly enhances the prognostic significance of CD8+ T cell infiltration in CRC. Taken together these data suggest that a cross-talk of potentially high clinical relevance takes place in CRC. Indeed, immunofluorescence staining of tissue sections from CRC showing evidence of infiltration by lymphocytes and granulocytes indicates that CD8+ and CD66b+ cells are frequently co-localized. Still unclear is whether neutrophils are able to modulate T-cell responsiveness to antigenic challenges (3;8). On the other hand activated T cells have previously been shown to enhance granulocyte survival and functions by the release of cytokines, including TNF-α, IFN-γ and GM-CSF (8). In additional studies I have extensively characterized phenotypic profiles of CRC infiltrating granulocytes, as compared to granulocytes from peripheral blood or infiltrating healthy mucosa adjacent to CRC. Functional studies performed by using tumor-derived and peripheral blood neutrophils indicate that these cells are able to efficiently co-stimulate CD8+ T cell activation induced by T-cell receptor (TCR) triggering, as witnessed by increased expression of CD69 activation marker, proliferation and IFN-γ release. Furthermore, an expansion of cells expressing a “central memory” phenotypic profile was also observed in cultures stimulated in the presence of autologous neutrophils. Furthermore, importantly, malignant transformation in the intestinal mucosa is associated with early translocation of microorganisms from the gut lumen (9). A variety of bacterial strains including Bacteroides fragilis and Fusobacterium nucleatum have been found to be highly represented in CRC (10). Capitalizing on these data, I have investigated the potential significance of the interaction with bacterial strains associated with CRC in the elicitation of the anti-tumor effects of neutrophils. I found that interaction with Fusobacterium nucleatum rapidly induces apoptosis in neutrophils and abrogates their co-stimulatory capacity. Taken together, my results contribute to the identification of CRC microenvironment as typically characterized by a “ménage à trois”, including cancerous cells, the immune system and gut colonizing microorganisms. The nature of the reciprocal interaction of these actors and its outcome are likely to decisively impact on CRC development and progressio

Identification of TPM2 and CNN1 as Novel Prognostic Markers in Functionally Characterized Human Colon Cancer-Associated Stromal Cells

Stromal infiltration is associated with poor prognosis in human colon cancers. However, the high heterogeneity of human tumor-associated stromal cells (TASCs) hampers a clear identification of specific markers of prognostic relevance. To address these issues, we established short-term cultures of TASCs and matched healthy mucosa-associated stromal cells (MASCs) from human primary colon cancers and, upon characterization of their phenotypic and functional profiles in vitro and in vivo, we identified differentially expressed markers by proteomic analysis and evaluated their prognostic significance. TASCs were characterized by higher proliferation and differentiation potential, and enhanced expression of mesenchymal stem cell markers, as compared to MASCs. TASC triggered epithelial-mesenchymal transition (EMT) in tumor cells in vitro and promoted their metastatic spread in vivo, as assessed in an orthotopic mouse model. Proteomic analysis of matched TASCs and MASCs identified a panel of markers preferentially expressed in TASCs. The expression of genes encoding two of them, calponin 1 (CNN1) and tropomyosin beta chain isoform 2 (TPM2), was significantly associated with poor outcome in independent databases and outperformed the prognostic significance of currently proposed TASC markers. The newly identified markers may improve prognostication of primary colon cancers and identification of patients at risk

Leaf extract from Mount Amiata chestnut as a novel anti-photoaging and skin care agent

Modern cosmetic industry is approaching medicinal plants with ethnobotanical tradition and pre-validated use (1). We studied the Italian chestnut (Castanea sativa Mill., Fagaceae) from Monte Amiata's forests, Tuscany (2), focusing on its byproducts, such as leaves. In different Italian regions, fresh leaves of chestnut have a long history of use by local people for dermatologic and cosmetic applications: e.g. as external poultice against sores in Tuscany (3), to make washes for skin diseases and inflammations in Sardinia and Calabria (4, 5), as infusion against dandruff in Liguria (6) and in the Abruzzo, Lazio and Molise National Park (7). In addition, the inhibitory activity against Staphylococcus aureus of refined European chestnut leaf extracts has been reported, with possible applications for treatment of skin and soft tissue infections (8). Driven by recent studies reported in literature, suggesting a scavenger and antioxidant activity of chestnut leaf extracts (9), our experimental protocol was focused on a 75% V/V ethanol extract obtained from Mount Amiata chestnut leaves, proposing its possible topical application as functional product in skin aging. Antioxidant and antiradical agents are, in fact, a useful strategy for the prevention of skin photoaging and oxidative stress-mediated skin diseases (10). C. sativa leaf extract contains flavonoids and hydroxycinnamic derivatives, with hyperoside being the most abundant constituent of the extract. Biological tests conducted on human keratinocytes showed that the extract protects cells from chemical (hydrogen peroxide) and physical (UVA irradiation) oxidative damage. The extract activity seems to be primarily related to free-radical scavenging, since cell levels of malondialdehyde, carbonylated proteins and reactive oxygen species decreased when cells were treated with 0.1% V/V extract, while superoxide dismutase activity and Nrf-2 mRNA expression were not affected by the extract at the same concentration. The extract, incorporated in an oil/water emulsion exhibited sun protection factor booster activity. Given these results, the Mount Amiata chestnut leaf extract could be an efficient opportunity in the treatment of extrinsic aging, in which one of the main targets is the neutralization of free radicals

Hemidesmus indicus induces immunogenic death in human colorectal cancer cells

The ability of anticancer treatments to promote the activation of tumor-reactive adaptive immune responses is emerging as a critical requirement underlying their clinical effectiveness. We investigated the ability of Hemidesmus indicus, a promising anticancer botanical drug, to stimulate immunogenic cell death in a human colorectal cancer cell line (DLD1). Here we show that Hemidesmus treatment induces tumor cell cytotoxicity characterized by surface expression of calreticulin, increased HSP70 expression and release of ATP and HMGB1. Remarkably, the exposure to released ICD-inducer factors from Hemidesmus-treated DLD1 cells caused a modest induction of CD14-derived dendritic cells maturation, as demonstrated by the increased expression of CD83. Moreover, at sub-toxic concentrations, H.i. treatment of monocytes and dendritic cells induced their mild activation, suggesting its additional direct immunostimulatory activity. These data indicate that Hemidesmus indicus induces immunogenic cell death in human tumor cells and suggest its potential relevance in innovative cancer immunotherapy protocols

Decoding of the surfaceome and endocytome in primary glioblastoma cells identifies potential target antigens in the hypoxic tumor niche

Abstract Immunotherapies with antibody–drug-conjugates (ADC) and CAR-T cells, targeted at tumor surface antigens (surfaceome), currently revolutionize clinical oncology. However, target identification warrants a better understanding of the surfaceome and how it is modulated by the tumor microenvironment. Here, we decode the surfaceome and endocytome and its remodeling by hypoxic stress in glioblastoma (GBM), the most common and aggressive brain tumor in adults. We employed a comprehensive approach for global and dynamic profiling of the surfaceome and endocytosed (endocytome) proteins and their regulation by hypoxia in patient-derived GBM cultures. We found a heterogeneous surface-endocytome profile and a divergent response to hypoxia across GBM cultures. We provide a quantitative ranking of more than 600 surface resident and endocytosed proteins, and their regulation by hypoxia, serving as a resource to the cancer research community. As proof-of-concept, the established target antigen CD44 was identified as a commonly and abundantly expressed surface protein with high endocytic activity. Among hypoxia induced proteins, we reveal CXADR, CD47, CD81, BSG, and FXYD6 as potential targets of the stressed GBM niche. We could validate these findings by immunofluorescence analyses in patient tumors and by increased expression in the hypoxic core of GBM spheroids. Selected candidates were finally confronted by treatment studies, showing their high capacity for internalization and ADC delivery. Importantly, we highlight the limited correlation between transcriptomics and proteomics, emphasizing the critical role of membrane protein enrichment strategies and quantitative mass spectrometry. Our findings provide a comprehensive understanding of the surface-endocytome and its remodeling by hypoxia in GBM as a resource for exploration of targets for immunotherapeutic approaches in GBM

The Irradiated brain microenvironment supports glioma stemness and survival via astrocyte-derived transglutaminase 2

The tumor microenvironment plays an essential role in supporting glioma stemness and radioresistance. Following radiotherapy, recurrent gliomas form in an irradiated microenvironment. Here we report that astrocytes, when pre-irradiated, increase stemness and survival of cocultured glioma cells. Tumor-naïve brains increased reactive astrocytes in response to radiation, and mice subjected to radiation prior to implantation of glioma cells developed more aggressive tumors. Extracellular matrix derived from irradiated astrocytes were found to be a major driver of this phenotype and astrocyte-derived transglutaminase 2 (TGM2) was identified as a promoter of glioma stemness and radioresistance. TGM2 levels increased after radiation in vivo and in recurrent human glioma, and TGM2 inhibitors abrogated glioma stemness and survival. These data suggest that irradiation of the brain results in the formation of a tumor-supportive microenvironment. Therapeutic targeting of radiation-induced, astrocyte-derived extracellular matrix proteins may enhance the efficacy of standard-of-care radiotherapy by reducing stemness in glioma

Landscape of surfaceome and endocytome in human glioma is divergent and depends on cellular spatial organization

Therapeutic strategies directed at the tumor surfaceome (TS), including checkpoint inhibitor blocking antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cells, provide a new armament to fight cancer. However, a remaining bottleneck is the lack of strategies to comprehensively interrogate patient tumors for potential TS targets. Here, we have developed a platform (tumor surfaceome mapping [TS-MAP]) integrated with a newly curated TS classifier (SURFME) that allows profiling of primary 3D cultures and intact patient glioma tumors with preserved tissue architecture. Moreover, TS-MAP specifically identifies proteins capable of endocytosis as tractable targets for ADCs and other modalities requiring toxic payload internalization. In high-grade gliomas that remain among the most aggressive forms of cancer, we show that cellular spatial organization (2D vs. 3D) fundamentally transforms the surfaceome and endocytome (e.g., integrins, proteoglycans, semaphorins, and cancer stem cell markers) with general implications for target screening approaches, as exemplified by an ADC targeting EGFR. The TS-MAP platform was further applied to profile the surfaceome and endocytome landscape in a cohort of freshly resected gliomas. We found a highly diverse TS repertoire between patient tumors, not directly associated with grade and histology, which highlights the need for individualized approaches. Our data provide additional layers of understanding fundamental to the future development of immunotherapy strategies, as well as procedures for proteomics-based target identification and selection. The TS-MAP platform should be widely applicable in efforts aiming at a better understanding of how to harness the TS for personalized immunotherapy

The Interplay Between Neutrophils and CD8+ T Cells Improves Survival in Human Colorectal Cancer

Purpose: Tumor infiltration by different T lymphocyte subsets is known to be associated with favorable prognosis in colorectal cancer. Still debated is the role of innate immune system. We investigated clinical relevance, phenotypes, and functional features of colorectal cancer-infiltrating CD66b+ neutrophils and their crosstalk with CD8+ T cells.Experimental Design: CD66b+ and CD8+ cell infiltration was analyzed by IHC on a tissue microarray including <650 evaluable colorectal cancer samples. Phenotypic profiles of tissue-infiltrating and peripheral blood CD66b+ cells were evaluated by flow cytometry. CD66b+/CD8+ cells crosstalk was investigated by in vitro experiments.Results: CD66b+ cell infiltration in colorectal cancer is significantly associated with increased survival. Interestingly, neutrophils frequently colocalize with CD8+ T cells in colorectal cancer. Functional studies indicate that although neutrophils are devoid of direct antitumor potential, coculture with peripheral blood or tumor-associated neutrophils (TAN) enhances CD8+ T-cell activation, proliferation, and cytokine release induced by suboptimal concentrations of anti-CD3 mAb. Moreover, under optimal activation conditions, CD8+ cell stimulation in the presence of CD66b+ cells results in increasing numbers of cells expressing CD45RO/CD62L "central memory" phenotype. Importantly, combined tumor infiltration by CD66b+ and CD8+ T lymphocytes is associated with significantly better prognosis, as compared with CD8+ T-cell infiltration alone.Conclusions: Neutrophils enhance the responsiveness of CD8+ T cells to T-cell receptor triggering. Accordingly, infiltration by neutrophils enhances the prognostic significance of colorectal cancer infiltration by CD8+ T cells, suggesting that they might effectively promote antitumor immunity. Clin Cancer Res; 1-12. (c)2017 AACR

Identification of TPM2 and CNN1 as Novel Prognostic Markers in Functionally Characterized Human Colon Cancer-Associated Stromal Cells

Stromal infiltration is associated with poor prognosis in human colon cancers. However, the high heterogeneity of human tumor-associated stromal cells (TASCs) hampers a clear identification of specific markers of prognostic relevance. To address these issues, we established short-term cultures of TASCs and matched healthy mucosa-associated stromal cells (MASCs) from human primary colon cancers and, upon characterization of their phenotypic and functional profiles in vitro and in vivo, we identified differentially expressed markers by proteomic analysis and evaluated their prognostic significance. TASCs were characterized by higher proliferation and differentiation potential, and enhanced expression of mesenchymal stem cell markers, as compared to MASCs. TASC triggered epithelial–mesenchymal transition (EMT) in tumor cells in vitro and promoted their metastatic spread in vivo, as assessed in an orthotopic mouse model. Proteomic analysis of matched TASCs and MASCs identified a panel of markers preferentially expressed in TASCs. The expression of genes encoding two of them, calponin 1 (CNN1) and tropomyosin beta chain isoform 2 (TPM2), was significantly associated with poor outcome in independent databases and outperformed the prognostic significance of currently proposed TASC markers. The newly identified markers may improve prognostication of primary colon cancers and identification of patients at risk

Additional file 1 of Decoding of the surfaceome and endocytome in primary glioblastoma cells identifies potential target antigens in the hypoxic tumor niche

Additional file 1: Supplementary Figures