Silvio Däster

CD133+, CD166+CD44+, and CD24+CD44+ Phenotypes Fail to Reliably Identify Cell Populations with Cancer Stem Cell Functional Features in Established Human Colorectal Cancer Cell Lines

Increasing evidence that cancers originate from small populations of so‐called cancer stem cells (CSCs), capable of surviving conventional chemotherapies and regenerating the original tumor, urges the development of novel CSC‐targeted treatments. Screening of new anticancer compounds is conventionally conducted on established tumor cell lines, providing sufficient material for high‐throughput studies. Whether tumor cell lines might comprise CSC populations resembling those of primary tumors, however, remains highly debated. We have analyzed the expression of defined phenotypic profiles, including CD133+, CD166+CD44+, and CD24+CD44+, reported as CSC‐specific in human primary colorectal cancer (CRC), on a panel of 10 established CRC cell lines and evaluated their correlation with CSC properties. None of the putative CSC phenotypes consistently correlated with stem cell‐like features, including spheroid formation ability, clonogenicity, aldehyde dehydrogenase‐1 activity, and side population phenotype. Importantly, CRC cells expressing putative CSC markers did not exhibit increased survival when treated with chemotherapeutic drugs in vitro or display higher tumorigenicity in vivo. Thus, the expression of CD133 or the coexpression of CD166/CD44 or CD24/CD44 did not appear to reliably identify CSC populations in established CRC cell lines. Our findings question the suitability of cell lines for the screening of CSC‐specific therapies and underline the urgency of developing novel platforms for anticancer drug discovery.

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; 23(14); 3847–58. ©2017 AACR.

Nuclear Expression of Snail Is an Independent Negative Prognostic Factor in Human Breast Cancer

Background. Snail is a key regulator of epithelial-mesenchymal transition of tumor cells. Several studies have shown nuclear Snail expression to be a negative prognostic factor in human cancer, where it is generally associated with more aggressive tumor behavior and worse survival. Objectives and Methods. To further explore the role of Snail expression in breast cancer, we conducted a study on a tissue microarray, encompassing 1043 breast cancer cases. Results. A total of 265 (25.4%) breast cancers were positive for Snail. Snail expression was significantly associated with greater tumor size, higher tumor stage and grade, positive lymph node status, and hormone receptor negative status and was differently expressed in the intrinsic subtypes of breast cancer, being the highest in the basal-like subtype and the lowest in the luminal A subtype. In multivariate analysis, Snail proved to be an independent negative prognostic factor for OS. In the intrinsic subtypes, Snail expression was a negative prognostic factor for OS in the luminal B HER2−, the luminal B HER2+, and the basal-like subtype. Conclusions. This is the first study demonstrating that nuclear Snail expression is an independent negative predictor of prognosis in breast cancer, thus suggesting that it may represent a potential therapeutic target.

Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment

Culture of cancerous cells in standard monolayer conditions poorly mirrors growth in three-dimensional architectures typically observed in a wide majority of cancers of different histological origin. Multicellular tumor spheroid (MCTS) culture models were developed to mimic these features. However, in vivo tumor growth is also characterized by the presence of ischemic and necrotic areas generated by oxygenation gradients and differential access to nutrients. Hypoxia and necrosis play key roles in tumor progression and resistance to treatment. To provide in vitro models recapitulating these events in highly controlled and standardized conditions, we have generated colorectal cancer (CRC) cell spheroids of different sizes and analyzed their gene expression profiles and sensitivity to treatment with 5FU, currently used in therapeutic protocols. Here we identify three MCTS stages, corresponding to defined spheroid sizes, characterized by normoxia, hypoxia, and hypoxia plus necrosis, respectively. Importantly, we show that MCTS including both hypoxic and necrotic areas most closely mimic gene expression profiles of in vivo-developing tumors and display the highest resistance to 5FU. Taken together, our data indicate that MCTS may mimic in vitro generation of ischemic and necrotic areas in highly standardized and controlled conditions, thereby qualifying as relevant models for drug screening purposes.

Gut microbiota modulate T cell trafficking into human colorectal cancer

Objective Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers. Design Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing. Results CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival. Conclusions Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues.

Absence of myeloperoxidase and CD8 positive cells in colorectal cancer infiltrates identifies patients with severe prognosis

Colorectal cancer (CRC) infiltration by cells expressing myeloperoxidase (MPO) or CD8 positive T lymphocytes has been shown to be independently associated with favorable prognosis. We explored the relationship occurring between CD8+ and MPO+ cell CRC infiltration, its impact on clinical-pathological features and its prognostic significance in a tissue microarray (TMA) including 1,162 CRC. We observed that CRC showing high MPO+ cell infiltration are characterized by a prognosis as favorable as that of cancers with high CD8+ T cell infiltration. However, MPO+ and CD8+ CRC infiltrating cells did not synergize in determining a more favorable outcome, as compared with cancers showing MPOhigh/CD8low or MPOlow/CD8high infiltrates. Most importantly, we identified a subgroup of CRC with MPOlow/CD8low tumor infiltration characterized by a particularly severe prognosis. Intriguingly, although MPO+ and CD8+ cells did not co-localize in CRC infiltrates, an increased expression of TIA-1 and granzyme-B was detectable in T cells infiltrating CRC with high MPO+ cell density.

OX40 expression enhances the prognostic significance of CD8 positive lymphocyte infiltration in colorectal cancer

Background OX40 is a TNF receptor family member expressed by activated T cells. Its triggering by OX40 ligand promotes lymphocyte survival and memory generation. Anti-OX40 agonistic monoclonal antibodies (mAb) are currently being tested in cancer immunotherapy. We explored the prognostic significance of tumor infiltration by OX40+ cells in a large colorectal cancer (CRC) collective. Methods OX40 gene expression was analyzed in 50 freshly excised CRC and corresponding healthy mucosa by qRT-PCR. A tissue microarray including 657 clinically annotated CRC specimens was stained with anti-OX40, -CD8 and -FOXP3 mAbs by standard immunohistochemistry. The CRC cohort was randomly split into training and validation sets. Correlations between CRC infiltration by OX40+ cells alone, or in combination with CD8+ or FOXP3+ cells, and clinical-pathological data and overall survival were comparatively evaluated. Results OX40 gene expression in CRC significantly correlated with FOXP3 and CD8 gene expression. High CRC infiltration by OX40+ cells was significantly associated with favorable prognosis in training and validation sets in univariate, but not multivariate, Cox regression analysis. CRC with OX40high/CD8high infiltration were characterized by significantly prolonged overall survival, as compared to tumors with OX40low/CD8high, OX40high/CD8low or OX40low/CD8low infiltration in both uni- and multivariate analysis. In contrast, prognostic significance of OX40+ and FOXP3+ cell infiltration was not enhanced by a combined evaluation. Irrespective of TNM stage, CRC with OX40high/CD8high density infiltrates showed an overall survival similar to that of all stage I CRC included in the study. Conclusions OX40high/CD8high density tumor infiltration represents an independent, favorable, prognostic marker in CRC with an overall survival similar to stage I cancers.

Two similar cases of internal hernia after laparoscopic Roux-en-Y gastric bypass surgery

Internal hernia after laparoscopic Roux-en-Y gastric bypass (LRYGB) surgery can lead to acute small bowel obstruction or chronic recurrent abdominal pain. We present two cases of internal hernias after antecolic antegastric LRYGB. Both patients presented to the emergency room with acute diffuse abdominal pain. Other than that, a physical examination and routine laboratory workup did not reveal any pathological finding. An abdominal CT was performed in both cases. It showed mesenteric torsion as a sign of internal hernia in one case, but remained inconclusive in the other patient. Immediate diagnostic laparoscopy was performed in both cases. Intraoperatively, both patients revealed an internal hernia, where the common channel herniated through the mesojejunal space. The conversion to upper median minilaparotomy was necessary for hernia reduction in both cases. No bowel resection was required and both patients recovered fully.

Massive surgical emphysema after perineal proctosigmoidectomy

An 83-year-old woman underwent an elective perineal proctosigmoidectomy (Altemeier procedure) for a rectal prolapse. On postoperative day 1, the patient presented with impressive subcutaneous emphysema involving the chest, neck and face without any other symptoms. A CT scan showed free air in the retroperitoneum, the intraperitoneal cavity, the mediastinum and a subcutaneous emphysema of the neck and the face. Air was also found around the coloanal anastomosis and an anastomotic leak was proven by rectal contrast agent. In this situation, a rectoscopy followed by a laparoscopy were immediately performed. The leak could not be visualised. Peritoneal lavage and drainage, followed by protective sigmoidostomy were carried out. After surgery, the follow-up was uneventful except a persistent but asymptomatic leak with a presacral cavity. The coloanal dehiscence was later proven in rectoscopy. Although sutured, it is still present and colostomy closure will eventually be possible in a few months.

Abstract 4031: Different expression of programmed death 1 (PD1) and its ligand (PD-L1) in esophageal and gastric cancer

Background: Prognosis of gastric and esophageal cancer remains poor. Improvements in gastric and esophageal cancer treatments are urgently needed. Programmed cell death 1 receptor (PD1) and its ligand 1 (PD-L1) are known to interact with T cells promoting epithelial cancer tolerance. Several therapeutic monoclonal antibodies blocking this interaction are reaching the clinical praxis. Therefore it is relevant to investigate the role of these biomarkers in different types of malignancy. Methods: Four different copies of tissue microarrays (TMA), each including healthy mucosa (n = 74) and a total of 241 clinically annotated malignancies of the esophagus (n = 80) and stomach (n = 161) were constructed and stained with PD1, PD-L1, and CD8 specific reagents. Results: Interestingly, only two cancer samples out of the 241 specimens investigated weakly expressed PD-L1. None of the normal mucosa epithelial cells expressed PD-L1. Stromal PD1 expression correlated with infiltration by CD8+ lymphocytes (rho = 0.4; P Conclusion: In contrast to other epithelial cancers, PD-L1 expression was virtually absent in the investigated malignancies of the esophagus and stomach. PD1 expression in the stroma surrounding such malignancies correlated with intraepithelial presence of T-cells CD8+ expression, and with better prognosis in gastric cancer patients. Citation Format: Silvio Daster, Serenella Eppenberger-Castori, Raoul A. Droeser, Hannah M. Schaefer, Giulio C. Spagnoli, Luigi Terracciano, Luigi Tornillo, Urs von Holzen. Different expression of programmed death 1 (PD1) and its ligand (PD-L1) in esophageal and gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4031.