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Supplemental Material, Supplemental_tables for Tumor Infiltration by OX40+ Cells Enhances the Prognostic Significance of CD16+ Cell Infiltration in Colorectal Cancer by Fabian Haak, Isabelle Obrecht, Nadia Tosti, Benjamin Weixler, Robert Mechera, Silvio Däster, Marco von Strauss, Tarik Delko, Giulio C. Spagnoli, Luigi Terracciano, Giuseppe Sconocchia, Markus von Flüe, Marko Kraljević and Raoul A. Droeser in Cancer Control

Aims. A trend towards local excision of early rectal cancers has prompted us to investigate if immunoprofiling might help in predicting lymph node involvement in this subgroup.Methods. A tissue microarray of 126 biopsies of early rectal cancer (T1 and T2) was stained for several immunomarkers of the innate and the adaptive immune response. Patients’ survival and nodal status were analyzed and correlated with infiltration of the different immune cells.Results. Of all tested markers, only CD8 (P=0.005) and TIA-1 (P=0.05) were significantly more frequently detectable in early rectal cancer biopsies of node negative as compared to node positive patients. Although these two immunomarkers did not display prognostic effect “per se,” CD8+ and, marginally, TIA-1 T cell infiltration could predict nodal involvement in univariate logistic regression analysis (OR 0.994; 95% CI 0.992–0.996;P=0.009and OR 0.988; 95% CI 0.984–0.994;P=0.05, resp.). An algorithm significantly predicting the nodal status in early rectal cancer based on CD8 together with vascular invasion and tumor border configuration could be calculated (P<0.00001).Conclusion. Our data indicate that in early rectal cancers absence of CD8+ T-cell infiltration helps in predicting patients’ nodal involvement.

Recent studies indicate that ER/PR/HER-2-negative (triple-negative, TN) breast cancers may be "CTA-rich" tumors, suggesting the possibility of CTA-based cancer vaccines as a treatment option for patients bearing these tumors. MAGE-A10 together with NY-ESO-1 is probably the most immunogenic CTA, representing a potentially highly attractive target of active specific immunotherapies. Paraffin-embedded tumor sections were collected retrospectively from 165 breast cancer patients diagnosed between 2002 and 2003. Immunohistochemical staining for MAGE-A10 and NY-ESO-1 was performed. The expression of MAGE-A10 and NY-ESO-1 was correlated with other clinicopathological variables. MAGE-A10 expression (score ≥ 2+) was detected in 105/164 (64%), and NY-ESO-1 expression (score ≥ 2+) was observed in 14/164 (8.5%) patients. No correlation between MAGE-A10 and NY-ESO-1 expression and tumor size, tumor grade, Ki-67 and lymph nodes status was detectable. MAGE-A10 expression was significantly associated with ER-negative (P = 0.002), PR-negative (P = 0.002) and HER-2-negative (P = 0.044) tumors. We clearly showed that MAGE-A10 is frequently expressed in the group of TN patients, where the majority (85.7%) of tumors express this CTA. Because of limited therapeutic options for the triple-negative breast cancer, the frequent expression of MAGE-A10 CTA in these cancers may offer the opportunity for a much needed additional treatment for this group of patients.

To evaluate MAGE tumor-associated antigen (TAA) expression in an extensive panel of normal and neoplastic tissues.TAAs of the MAGE family represent targets of active specific immunotherapy. Limited-size studies indicate that they are expressed in normal testis and tumors of different histologies. High-throughput tissue microarray (TMA) technology and MAGE TAA-specific monoclonal antibodies now allow us to comprehensively evaluate their expression in large numbers of tissues and to address clinical correlations.A TMA containing 3,520 samples from 197 different tissues and a non-small-cell lung cancer TMA including 301 specimens were stained using the MAGE TAA-specific monoclonal antibody 57B. For patients with squamous cell carcinoma of the lung, the dichotomous result (positive vs. negative) of MAGE TAA staining was used as a predictor variable along with other covariates in proportional hazard regression analysis of tumor-specific survival.MAGE TAAs are expressed with frequencies ranging between 22.7% (larynx) and 50% of cases (lung) in squamous cell carcinomas from different anatomic areas and in large cell carcinomas of the lung (37.9%). The authors provide here the first description of MAGE TAA expression in basalioma (48.1%). To investigate the clinical significance of MAGE expression in a frequently positive tumor type, a non-small-cell lung cancer, TMA was then studied. In this TMA 43.2% of tumors were 57B positive. In patients with squamous cell carcinoma, MAGE TAA positivity was significantly correlated with a shorter tumor-specific survival in the proportional hazard regression analysis model.These data suggest novel potential therapeutic indications in different types of cancers. In lung squamous cell carcinoma, the significant association of MAGE TAA expression with poor prognosis suggests that patients with 57B-positive tumors may benefit from early, specific immunotherapy procedures.

T cadherin (T cad) is gaining recognition as a determinant for the development of incipient invasive squamous cell carcinoma (SCC). However effects of T cad expression on the metastatic potential of SCC have not been studied. Here using a murine model of experimental metastasis following tail vein injection of A431 SCC cells we report that loss of T cad increases both the incidence and rate of appearance of lung metastases and reduces survival. T cad silenced SCC metastases are highly disordered with evidence of single cell dissemination away from main foci whereas SCC metastases overexpressing T cad develop as compact tightly organized sheets. SCC adhesion to vascular endothelial cells (EC) in culture and transmigration across cultures of vascular and lymphatic EC is increased for T cad silenced SCC and decreased for T cad overexpressing SCC. Confocal microscopy shows that T cad silenced SCC adherent on EC display an elongated morphology with long thin extensions and a high degree of intercalation within the EC monolayer whereas SCC overexpressing T cad formed poorly spread multicellular aggregates that rather remain on the outer surface of the EC monolayer. Our data suggest that loss of T cad can increase metastatic potential and aggressiveness of SCC possibly due to facilitating arrest and extravasation through the vascular wall and/or more efficient establishment of metastases in the new microenvironment.

// Silvio Daster 1, * , Nunzia Amatruda 1, 2, * , Diego Calabrese 2 , Robert Ivanek 2 , Eleonora Turrini 3 , Raoul A. Droeser 1 , Paul Zajac 1, 2 , Carmela Fimognari 3 , Giulio C. Spagnoli 1, 2 , Giandomenica Iezzi 1, 2 , Valentina Mele 1, 2, ** , Manuele G. Muraro 1, 2, ** 1 Department of Surgery, University Hospital Basel, Basel, Switzerland 2 Department of Biomedicine, University of Basel, Basel, Switzerland 3 Department for Life Quality Studies, University of Bologna, Rimini, Italy * These authors have contributed equally to this work ** Shared senior authorship Correspondence to: Valentina Mele, email: valentina.mele@usb.ch Manuele G. Muraro, email: manuele.muraro@usb.ch Keywords: multicellular tumor spheroids, three-dimensional culture, tumor model, hypoxia, necrosis Received: April 19, 2016 Accepted: September 19, 2016 Published: December 10, 2016 ABSTRACT 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.