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Supplementary Table 1 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Supplementary Table 2 from Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Inflammation and oxidative stress play a crucial role in the development of colorectal cancer (CRC) and interference with these mechanisms represents a strategy in CRC chemoprevention. Allopurinol, a safe molecular scavenger largely used as antigout agent, has been shown to increase survival of patients with advanced CRC and to reduce CRC incidence in long-term gout users in epidemiologic studies. We conducted a randomized, double-blind, placebo-controlled preoperative trial in subjects with colorectal adenomatous polyps to assess the activity of allopurinol on biomarkers of colorectal carcinogenesis.After complete colonoscopy and biopsy of the index polyp, 73 subjects with colorectal adenomas were assigned to either placebo or one of two doses of allopurinol (100 mg or 300 mg) and treated for four weeks before polyp removal. Change of Ki-67 labeling index in adenomatous tissue was the primary endpoint. Secondary endpoints were the immunohistochemical (IHC) expression of NF-κB, β-catenin, topoisomerase-II-α, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) in adenomatous polyps and normal adjacent colonic tissue.Compared with placebo, Ki-67 levels were not significantly modulated by allopurinol, whereas β-catenin and NF-κB expression levels decreased significantly in adenomatous tissue, with a mean change from baseline of −10.6%, 95% confidence interval (CI), −20.5 to −0.7, and −8.1%, 95% CI, −22.7 to 6.5, respectively. NF-κB also decreased significantly in normal adjacent tissue (−16.4%; 95% CI, −29.0 to −3.8). No dose–response relationship was noted, except for NF-κB expression in normal tissue.Allopurinol can inhibit biomarkers of oxidative activation in colon adenomatous polyps and normal adjacent tissue. Further studies should define its potential chemopreventive activity. Cancer Prev Res; 6(2); 74–81. ©2012 AACR.

Commentary on This Article from A Randomized, Placebo-Controlled, Preoperative Trial of Allopurinol in Subjects with Colorectal Adenoma

Purpose: The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor–resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies.Experimental Design: We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways.Results: Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor–resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents.Conclusions: This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.

Supplementary Figures S1-S2 from Vascular Endothelial Growth Factor Receptor-1 Contributes to Resistance to Anti–Epidermal Growth Factor Receptor Drugs in Human Cancer Cells

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Tumor-associated fibroblasts (TAF) exert immunosuppressive effects in colorectal carcinoma (CRC), impairing the recognition of tumor cells by effector lymphocytes, including Vδ2 T cells. Herein, we show that CRC-derived TAF can be turned by zoledronic acid (ZA), in soluble form or as antibody-drug conjugate (ADC), into efficient stimulators of Vδ2 T cells. CRC-TAF, obtained from patients, express the epidermal growth factor receptor (EGFR) and the butyrophilin family members BTN3A1/BTN2A1. These butyrophilins mediate the presentation of the phosphoantigens, accumulated in the cells due to ZA effect, to Vδ2 T cells. CRC-TAF exposed to soluble ZA acquired the ability to trigger the proliferation of Vδ2 T cells, in part represented by effector memory cells lacking CD45RA and CD27. In turn, expanded Vδ2 T cells exerted relevant cytotoxic activity towards CRC cells and CRC-TAF when primed with soluble ZA. Of note, also the ADC made of the anti-EGFR cetuximab (Cet) and ZA (Cet-ZA), that we recently described, induced the proliferation of anti-tumor Vδ2 T lymphocytes and their activation against CRC-TAF. These findings indicate that ZA can educate TAF to stimulate effector memory Vδ2 T cells; the Cet-ZA ADC formulation can lead to the precise delivery of ZA to EGFR+ cells, with a double targeting of TAF and tumor cells.

Castration-resistant prostate cancer (CRPC) is an incurable stage of the disease. A multivariate principal component analysis on CRPC in vitro models identified aspartyl (asparaginyl) β hydrolase (ASPH) as the most relevant molecule associated with the CRPC phenotype. ASPH is overexpressed in various malignant neoplasms and catalyzes the hydroxylation of aspartyl and asparaginyl residues in the epidermal growth factor (EGF)-like domains of proteins like NOTCH receptors and ligands, enhancing cell motility, invasion and metastatic spread. Bioinformatics analyses of ASPH in prostate cancer (PCa) and CRPC datasets indicate that ASPH gene alterations have prognostic value both in PCa and CRPC patients. In CRPC cells, inhibition of ASPH expression obtained through specific small interfering RNA or culturing cells in hypoxic conditions, reduced cell proliferation, invasion and cyclin D1 expression through modulation of the NOTCH signaling. ASPH and HIF1α crosstalk, within a hydroxylation-regulated signaling pathway, might be transiently driven by the oxidative stress evidenced inside CRPC cells. In addition, increased phosphorylation of GSK3β by ASPH silencing demonstrates that ASPH regulates GSK3β activity inhibiting its interactions with upstream kinases. These findings demonstrate the critical involvement of ASPH in CRPC development and may represent an attractive molecular target for therapy.

Immune checkpoint (IC) molecules act as receptors, expressed on immune effector cells, that are able to recognize specific ligands in normal or tumor cells [...]

Colorectal Cancer (CRC) is one of most frequent malignant cancers, showing high lethality worldwide [...]

About 15% of colorectal cancers (CRCs) are diagnosed as advanced, metastatic stage IV, a patient condition with an average survival of 2 [...]

The identification and validation of simple, reliable and reproducible three dimensional (3D) in vitro culture systems represent a major challenge in the field of anticancer drug development [...]

Fibroblasts are incredible cells [...]

Colorectal cancer (CRC) is a relatively slow-growing tumor that can be treated successfully when identified in the early stages [...]

Simple Summary The classical approach to study the immune response against a tumor was mixing immune cells with tumor cell suspensions in several experimental settings. These models lack the appropriate tissue architecture in which the immune response takes place and do not consider other cellular and extracellular players of the tumor microenvironment essential to understand the anti-tumor immune response. Thus, to confirm in vitro data, in vivo experiments have been extensively performed, using animal models that may not fully reproduce what happens in humans. Indeed, in animal-based studies, tumors are artificially generated in a short time, and immune cell subsets and receptor-ligands pairs, involved in tumor cells recognition by the immune system, are often different from human counterparts. To reduce the number of animals used, and possibly replace animal models, alternative methods of culture have been developed. Herein, some of these approaches will be described, highlighting their advantages and disadvantages, focusing on natural killer cells as the first line of anti-tumor effector cells able to contrast tumor growth. Abstract Several approaches have shown that the immune response against tumors strongly affects patients’ clinical outcome. Thus, the study of anti-tumor immunity is critical to understand and potentiate the mechanisms underlying the elimination of tumor cells. Natural killer (NK) cells are members of innate immunity and represent powerful anti-tumor effectors, able to eliminate tumor cells without a previous sensitization. Thus, the study of their involvement in anti-tumor responses is critical for clinical translation. This analysis has been performed in vitro, co-incubating NK with tumor cells and quantifying the cytotoxic activity of NK cells. In vivo confirmation has been applied to overcome the limits of in vitro testing, however, the innate immunity of mice and humans is different, leading to discrepancies. Different activating receptors on NK cells and counter-ligands on tumor cells are involved in the antitumor response, and innate immunity is strictly dependent on the specific microenvironment where it takes place. Thus, three-dimensional (3D) culture systems, where NK and tumor cells can interact in a tissue-like architecture, have been created. For example, tumor cell spheroids and primary organoids derived from several tumor types, have been used so far to analyze innate immune response, replacing animal models. Herein, we briefly introduce NK cells and analyze and discuss in detail the properties of 3D tumor culture systems and their use for the study of tumor cell interactions with NK cells.

Objective A comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC. Design Thirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence. Results We discovered that a previously unappreciated innate lymphocyte population (Lin–CD7+CD127–CD56+CD45RO+) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103+CD69+) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and γδ T cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated γδ T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδ T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes. Conclusion This work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.

Preclinical models for the definition of anti-cancer drug safety and efficacy are constantly evolving [...]

Colorectal cancer’s (CRC) ability to invade local tissues and lymph nodes and generate distant metastases is the key for TNM classification. Aspartate-β-hydroxylase (ASPH), a transmembrane protein that catalyzes Notch receptors and ligand activation, is involved in tumor invasion. Because Notch is involved in gut homeostasis, it could be a target for CRC therapy. ASPH mRNA and protein expression, promoter methylation and gene copy numbers were evaluated using the TCGA and CPTAC human CRC datasets. Using digital pathology, ASPH was scored in the luminal area (LM), center tumor (CT) and invasive margin (IM) of 100 human CRCs. The effect of ASPH targeting on invasiveness and viability was tested by siRNA knockdown and small molecule inhibitors (SMI). Bioinformatics analysis showed increased expression of ASPH mRNA and protein in CRC, paired with a decreased methylation profile. ASPH genetic gain or amplification was frequent (56%), while deletion was rare (0.03%). Digital pathology analysis showed that ASPH exerted its pathological activity in the invasive margin of the tumor, affecting invasive front morphology, tumor budding and patients’ overall survival. In vitro, ASPH targeting by siRNA or SMI reduced cell invasion and growth and caused Notch-1 downregulation. This study demonstrates that ASPH targeting by specific inhibitors could improve CRC treatment strategies.

Regenerative strategies for human articular cartilage are still challenging despite the presence of resident progenitor cell population. Today, many efforts in the field of regenerative medicine focus on the use of platelet derivatives due to their ability to reactivate endogenous mechanisms supporting tissue repair. While their use in orthopedics continues, mechanisms of action and efficacy need further characterization. We describe that the platelet lysate (PL) is able to activate chondro-progenitor cells in a terminally differentiated cartilage tissue. Primary cultures of human articular chondrocytes (ACs) and cartilage explants were set up from donor hip joint biopsies and were treated in vitro with PL. PL recruited a chondro-progenitors (CPCs)-enriched population from ex vivo cartilage culture, that showed high proliferation rate, clonogenicity and nestin expression. CPCs were positive for in vitro tri-lineage differentiation and formed hyaline cartilage-like tissue in vivo without hypertrophic fate. Moreover, the secretory profile of CPCs was analyzed, together with their migratory capabilities. Some CPC-features were also induced in PL-treated ACs compared to fetal bovine serum (FBS)-control ACs. PL treatment of human articular cartilage activates a stem cell niche responsive to injury. These facts can improve the PL therapeutic efficacy in cartilage applications.

Colorectal cancer’s (CRC) ability to invade local tissues and lymph nodes and generate distant metastases is the key for TNM classification. Aspartate-β-hydroxylase (ASPH), a transmembrane protein that catalyzes Notch receptors and ligand activation, is involved in tumor invasion. Because Notch is involved in gut homeostasis, it could be a target for CRC therapy. ASPH mRNA and protein expression, promoter methylation and gene copy numbers were evaluated using the TCGA and CPTAC human CRC datasets. Using digital pathology, ASPH was scored in the luminal area (LM), center tumor (CT) and invasive margin (IM) of 100 human CRCs. The effect of ASPH targeting on invasiveness and viability was tested by siRNA knockdown and small molecule inhibitors (SMI). Bioinformatics analysis showed increased expression of ASPH mRNA and protein in CRC, paired with a decreased methylation profile. ASPH genetic gain or amplification was frequent (56%), while deletion was rare (0.03%). Digital pathology analysis showed that ASPH exerted its pathological activity in the invasive margin of the tumor, affecting invasive front morphology, tumor budding and patients’ overall survival. In vitro, ASPH targeting by siRNA or SMI reduced cell invasion and growth and caused Notch-1 downregulation. This study demonstrates that ASPH targeting by specific inhibitors could improve CRC treatment strategies.

Observational/retrospective studies indicate that prostaglandin-endoperoxide synthase-2 (PTGS2) inhibitors could positively affect colorectal cancer (CRC) patients&rsquo, survival after diagnosis. To obtain an acceptable cost/benefit balance, the inclusion of PTGS2 inhibitors in the adjuvant setting needs a selective criterion. We quantified the 72 kDa, CRC-associated, glycosylated form of PTGS2 in 100 frozen CRC specimens and evaluated PTGS2 localization by IHC in the same tumors, scoring tumor epithelial-derived and stroma-derived fractions. We also investigated the involvement of interleukin-1 beta (IL1&beta, ) in PTGS2 induction, both in vitro and in CRC lysates. Finally, we used overall survival (OS) as a criterion for patient selection. Glycosylated PTGS2 can be quantified with high sensibility in tissue lysates, but the expression in both tumor and stromal cells limits its use for predictive purposes. Immunohistochemistry (IHC) analysis indicates that stromal PTGS2 expression could exert a protective role on patient OS. Stromal PTGS2 was prevalently expressed by cancer-associated fibroblasts exerting a barrier function near the gut lumen, and it apparently favored the antitumor M1 macrophage population. IL1&beta, was directly linked to gPTGS2 expression both in vitro and in tumors, but its activity was apparently prevalent on the stromal cell population. We suggest that stromal PTGS2 could exert a positive effect on patients OS when expressed in the luminal area of the tumor.

Aminobisphosphonates, such as zoledronic acid (ZA), have shown potential in the treatment of different malignancies, including colorectal carcinoma (CRC). Yet, their clinical exploitation is limited by their high bone affinity and modest bioavailability. Here, ZA is encapsulated into the aqueous core of spherical polymeric nanoparticles (SPNs), whose size and architecture resemble that of biological vesicles. On V&delta, 2 T cells, derived from the peripheral blood of healthy donors and CRC patients, ZA-SPNs induce proliferation and trigger activation up to three orders of magnitude more efficiently than soluble ZA. These activated V&delta, 2 T cells kill CRC cells and tumor spheroids, and are able to migrate toward CRC cells in a microfluidic system. Notably, ZA-SPNs can also stimulate the proliferation of V&delta, 2 T cells from the tumor-infiltrating lymphocytes of CRC patients and boost their cytotoxic activity against patients&rsquo, autologous tumor organoids. These data represent a first step toward the use of nanoformulated ZA for immunotherapy in CRC patients.

Inflammation plays a central role in prostate cancer (PCa) development through significant crosstalk between the COX-2-ErbB family receptor network and androgen receptor (AR)-EGFR signaling pathways. The purpose of this work was to determine the ability of the COX-2 inhibitor Celecoxib to modulate the EGFR-AR signaling pathway in androgen-dependent PCa cells and to provide a rationale for its beneficial use in chemopreventive strategies. Functional studies of Celecoxib activity were performed on LNCaP prostate cancer cells. Western blotting, gene expression analysis, dual-luciferase reporter assay and ELISA were applied to assess the Celecoxib mechanisms of action. We found that Celecoxib, through EGF and amphiregulin (AREG) induction, caused EGFR and ErbB2 activation and consequent degradation associated with the inhibition of androgenic signaling. By upregulating the E3 ubiquitin ligase Nrdp1, Celecoxib also efficiently downregulated ErbB3, which is strongly implicated in castration-resistant prostate cancer. Lastly, Celecoxib directly regulated AR transcription and translation independent of ErbB activation by downregulating the RNA binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K). The simultaneous suppression of ErbB kinases and androgen signaling by Celecoxib represents a novel strategy to interrupt the vicious cycle of AR/ErbB cross-talk with the primary purpose of undermining their resilient signaling in prostate cancer progression. Our data provide important premises for the chemopreventive use of Celecoxib in the clinical management of prostate cancer.

Background: Prostate cancer (PCa) is a significant health concern throughout the world. Standard therapy for advanced disease consists of anti-androgens, however, almost all prostate tumors become castration resistant (CRPC). Progression from androgen-sensitive PCa to CRPC is promoted by inflammatory signaling through cyclooxygenase-2 (COX-2) expression and ErbB family receptors/AKT activation, compensating androgen receptor inactivity. Methods: Making use of CRPC cell lines, we investigated the effects of the anti-inflammatory drug celecoxib. Biochemical data obtained using immunoblotting, enzyme-linked immunosorbent assay (ELISA), invasion, and xenografts were further integrated by bioinformatic analyses. Results: Celecoxib reduced cell growth and induced apoptosis through AKT blockade, cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), and proteasomal degradation of the anti-apoptotic protein Mcl-1. Epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 degradation, and heterogeneous nuclear ribonucleoprotein K (hnRNP K) downregulation, further amplified the inhibition of androgen signaling. Celecoxib reduced the invasive phenotype of CRPC cells by modulating NF-&kappa, B activity and reduced tumor growth in mice xenografts when administered in association with the anti-EGFR receptor antibody cetuximab. Bioinformatic analyses on human prostate cancer datasets support the relevance of these pathways in PCa progression. Conclusions: Signaling nodes at the intersection of pathways implicated in PCa progression are simultaneously modulated by celecoxib treatment. In combination therapies with cetuximab, celecoxib could represent a novel therapeutic strategy to curb signal transduction during CRPC progression.

Hypoxia and the accumulation of hypoxia-inducible factors (HIFs) in tumors have been associated with therapeutic resistance and with autophagy establishment. We examined the effects of stable knockdown of HIF-1&alpha, or HIF-2&alpha, expression on autophagy and drug resistance in colon cancer cells. We found that under normoxic conditions, malignant cells exhibit increased basal levels of autophagy, compared with non-malignant cells, in addition to the previously reported coexpression of HIF-1&alpha, and HIF-2&alpha, Knockdown of HIF-1&alpha, expression resulted in increased autophagic and apoptotic cell death, indicating that the survival of cells is HIF-dependent. Cytotoxic-induced cell death was significantly increased by knockdown of HIFs but not by autophagy inhibition. Strikingly, although malignancy-resistant cells were sensitized to death by nutrient stress, the combination with HIF-2&alpha, depletion, but not with HIF-1&alpha, depletion, induced severe cell death. Oxidative stress levels were significantly increased as a result of HIF-2&alpha, specific inhibition or silencing suggesting that this may contribute to sensitize cells to death. The in vitro results were confirmed in vivo using a xenograft mouse model. We found that coordinated autophagy and mTOR inhibition enhanced cell death and induced tumor remission only in HIF-2&alpha, silenced cells. Finally, using a specific HIF-2&alpha, inhibitor alone or in combination with drugs in patient-derived primary colon cancer cells, overcame their resistance to 5-FU or CCI-779, thus emphasizing the crucial role played by HIF-2&alpha, in promoting resistance and cell survival.

Due to their osteoconductive and inductive properties, a variety of calcium phosphate (CaP) scaffolds are commonly used in orthopaedics as graft material to heal bone defects. In this study, we have used two CaP scaffolds with different hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ratios (MBCP®; 60/40 and MBCP+ ®; 20/80) to investigate their intrinsic capacity to favour human bone marrow stem cells (hBMSCs) osteogenic differentiation capacity. We report that MBCP+ ® showed in in vitro culture model a higher rate of calcium ion release in comparison with MBCP®. In two defined coculture systems, the hBMSC seeded onto MBCP+ ® presented an increased amount of VEGF secretion, resulting in an enhanced endothelial cell proliferation and capillary formation compared with hBMSC seeded onto MBCP®. When both ceramics combined with hBMSC were implanted in a nude mouse model, we observed a faster osteogenic differentiation and enhancement mature bone deposition sustained by the presence of a vast host vasculature within the MBCP+ ® ceramics. Bone formation was observed in samples highly positive to the activation of calcium sensing receptor protein (CaSr) on the surface of seeded hBMSC that also shown higher BMP-2 protein expression. With these data we provide valuable insights in the possible mechanisms of ossification and angiogenesis by hBMSC that we believe to be primed by calcium ions released from CaP scaffolds. Evidences could lead to an optimization of ceramic scaffolds to prime bone repair.

It is well established that natural killer (NK) cells are involved in both innate and adaptive immunity. Indeed, they can recognize molecules induced at the cell surface by stress signals and virus infections. The functions of NK cells in the gut are much more complex. Gut NK cells are not precisely organized in lymphoid aggregates but rather scattered in the epithelium or in the stroma, where they come in contact with a multitude of antigens derived from commensal or pathogenic microorganisms in addition to components of microbiota. Furthermore, NK cells in the bowel interact with several cell types, including epithelial cells, fibroblasts, macrophages, dendritic cells, and T lymphocytes, and contribute to the maintenance of immune homeostasis and development of efficient immune responses. NK cells have a key role in the response to intestinal bacterial infections, primarily through production of IFNγ, which can stimulate recruitment of additional NK cells from peripheral blood leading to amplification of the anti-bacterial immune response. Additionally, NK cells can have a role in the pathogenesis of gut autoimmune inflammatory bowel diseases (IBDs), such as Crohn's Disease and Ulcerative Colitis. These diseases are considered relevant to the generation of gastrointestinal malignancies. Indeed, the role of gut-associated NK cells in the immune response to bowel cancers is known. Thus, in the gut immune system, NK cells play a dual role, participating in both physiological and pathogenic processes. In this review, we will analyze the known functions of NK cells in the gut mucosa both in health and disease, focusing on the cross-talk among bowel microenvironment, epithelial barrier integrity, microbiota, and NK cells.