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Background The role of ATF2 in colon cancer (CC) is controversial. Recently, we reported that low ATF2 expression is characteristic of highly invasive tumors, suggesting that ATF2 might also be involved in therapy resistance. 5-Fluorouracil (5-FU) is the best-known chemotherapeutic drug for CC, but drug resistance affects its curative effect. To date, the role of ATF2 in the 5-FU response remains elusive. Methods/Results For our study, we had available HCT116 cells (wild-type p53) and HT29 colon tumor cells (mutant p53) and their corresponding CRISPR‒Cas9-generated ATF2-KO clones. We observed that loss of ATF2 triggered dose- and time-dependent 5-FU resistance in HCT116 cells by activating the DNA damage response (DDR) pathway with high p-ATRThr1989 and p-Chk1Ser317 levels accompanied by an increase in the DNA damage marker γ-H2AX in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. Chk1 inhibitor studies causally displayed the link between DDR and drug resistance. There were contradictory findings in HT29 ATF2-KO cells upon 5-FU exposure with low p-Chk1Ser317 levels, strong apoptosis induction, but no effects on DNA damage. In ATF2-silenced HCT116 p53−/− cells, 5-FU did not activate the DDR pathway. Co-immunoprecipitation and proximity ligation assays revealed that upon 5-FU treatment, ATF2 binds to ATR to prevent Chk1 phosphorylation. Indeed, in silico modelling showed reduced ATR-Chk1 binding when ATF2 was docked into the complex. Conclusions We demonstrated a novel ATF2 scaffold function involved in the DDR pathway. ATF2-negative cells are highly resistant due to effective ATR/Chk1 DNA damage repair. Mutant p53 seems to overwrite the tumor suppressor function of ATF2.

Neoplasms of the lungs are the leading cause of cancer incidence and mortality worldwide. Although immunotherapy has increased the overall survival of patients with lung cancer, there is the need to improve this treatment. At this regard, blood lipid levels are thought to be linked to cancer risk and thus a preventive intervention through regulation of the nutrition of patients with lung cancer is gaining much attention. In this study, we therefore asked about the contribution of serum lipids and cholesterol cellular metabolism in lung cancer development and progression. We measured different serum lipids and analyzed cholesterol synthesis enzymes 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and acetyl-coenzyme A cholesterol acetyltransferase 1 (ACAT1) as well as the cholesterol cellular export protein ATP-binding cassette (ABC) A-1 mRNA by quantitative PCR (qPCR) in the control and tumoral regions of post-surgery lung tissues to analyze the accumulation of cholesterol in cancer cells in a cohort of patients with lung adenocarcinoma (LUAD). We found that triglycerides in serum directly correlated with the body mass index (BMI) in patients with LUAD. By contrast, we found that high-density lipoprotein (HDL) cholesterol inversely correlated with the BMI, C-reactive protein (CRP) and overall survival and total cholesterol inversely correlated with the tumor diameter, serum CRP and overall survival in these LUAD patients. Functionally, the role of cholesterol is indispensable for the growth and development of normal animal cells where it is tightly regulated. Excess of cellular cholesterol regulated by HMGCR is converted to cholesteryl esters by the enzyme ACAT1 and exported extracellularly by the cholesterol transporter ABCA1. Here we found HMGCR and ACAT1 upregulated and ABCA1 downregulated in the lung’s tumoral region of our LUAD cohort, indicating cholesterol dysregulated cellular export in lung tumor cells.

Clinico-pathological features of analyzed cohorts and subgroups.

Distribution of stromal tumor infiltrating lymphocytes in analyzed cohorts and subgroups.

Figure S1. Selected images of different sTILs amounts, illustration of applied scoring method and images of IHC stainings. Figure S2. TLS in MIBC Figure S3. Subtyping of the CCC-EMN cohort and the TCGA-Cohort according to the MDACC-subtyping gene signature Figure S4. TCGA-Basal tumors (n=142). CIBERSORT and Mutational Burden. Figure S5. TCGA Luminal tumors (n=243). CIBERSORT and Mutational Burden. Figure S6. Distribution of tumor mutational burden (TMB; based on total single nucleotide variants) and neoantigens among different cut-offs of stromal TILs. Figure S7. TCGA - Antigen presenting cell signature (AGPS) and combined adjuvant platinum-containing chemotherapy cohort.

CIBERSORT - Spearman rank correlations between predicted immune cell populations in the TCGA cohort.

Inflammation gene signature TCGA: Full table of included genes.

Antigen presenting cell signature TCGA: Full table of included genes.

Multivariable Survival Models.

Reference Sequences - Nanostring analysis for subtyping (CCC-EMN cohort).

Supplementary Figure 3: Representative dot plots of NFATc1 levels in T cells of lung tumor-bearing B6 WT mice in vivo treated and in vitro re-challenged with αPD-1antibodies.

Decreased NFATc1 in T cells in the tumoural region of NSCLC patients with advanced disease stages.Cytokines secreted from NFATc1Î"CD4 total lung cells did not influence lung tumour cell growth. Representative dot plots of NFATc1 levels in T cells of lung tumor-bearing B6 WT mice in vivo treated and in vitro re-challenged with αPD-1antibodies.

Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor activated by T-cell receptor (TCR) and Ca2+ signaling that affects T-cell activation and effector function. Upon tumor antigen challenge, TCR and calcium-release–activated channels are induced, promoting NFAT dephosphorylation and translocation into the nucleus. In this study, we report a progressive decrease of NFATc1 in lung tumor tissue and in tumor-infiltrating lymphocytes (TIL) of patients suffering from advanced-stage non–small cell lung cancer (NSCLC). Mice harboring conditionally inactivated NFATc1 in T cells (NFATc1ΔCD4) showed increased lung tumor growth associated with impaired T-cell activation and function. Furthermore, in the absence of NFATc1, reduced IL2 influenced the development of memory CD8+ T cells. We found a reduction of effector memory and CD103+ tissue-resident memory (TRM) T cells in the lung of tumor-bearing NFATc1ΔCD4 mice, underlining an impaired cytotoxic T-cell response and a reduced TRM tissue-homing capacity. In CD4+ICOS+ T cells, programmed cell death 1 (PD-1) was induced in the draining lymph nodes of these mice and associated with lung tumor cell growth. Targeting PD-1 resulted in NFATc1 induction in CD4+ and CD8+ T cells in tumor-bearing mice and was associated with increased antitumor cytotoxic functions. This study reveals a role of NFATc1 in the activation and cytotoxic functions of T cells, in the development of memory CD8+ T-cell subsets, and in the regulation of T-cell exhaustion. These data underline the indispensability of NFATc1 for successful antitumor immune responses in patients with NSCLC.Significance: The multifaceted role of NFATc1 in the activation and function of T cells during lung cancer development makes it a critical participant in antitumor immune responses in patients with NSCLC. Cancer Res; 78(13); 3619–33. ©2018 AACR.

Supplementary Figure 2: Cytokines secreted from NFATc1Î"CD4 total lung cells did not influence lung tumour cell growth.

Supplementary Figure 1: Decreased NFATc1 in T cells in the tumoural region of NSCLC patients with advanced disease stages.

The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specificZeb1deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers is reduced upon fibroblast-specific loss ofZeb1. Single-cell transcriptomics, histological andin vitrocharacterization reveal a crucial role in CAF polarization, promoting myofibroblastic features whilst restricting inflammatory activation.Zeb1deficiency impairs collagen deposition and CAF barrier function but increases cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, theZeb1-deficient CAF repertoire sensitizes to immune checkpoint inhibition, pointing to a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.

Simple Summary A lower tumor–stroma ratio within a tumor correlates with a poorer outcome, i.e., with a higher risk of death. The assessment of this ratio by humans is prone to errors, and when presented the same case, the ratios reported by multiple pathologists will oftentimes deviate significantly. The aim of our work was to predict the tumor–stroma ratio automatically using deep neural segmentation networks. The assessment comprises two steps: recognizing the different tissue types and estimating their ratio. We compared both steps individually to human observers and showed that (i) the outlined automatic method yields good segmentation results and (ii) that human estimations are consistently higher than the automated estimation and deviate significantly for a hand-annotated ground truth. We showed that including an additional evaluation step for our segmentation results and relating the segmentation quality to deviations in tumor–stroma assessment provides helpful insights. Abstract The tumor–stroma ratio (TSR) has been repeatedly shown to be a prognostic factor for survival prediction of different cancer types. However, an objective and reliable determination of the tumor–stroma ratio remains challenging. We present an easily adaptable deep learning model for accurately segmenting tumor regions in hematoxylin and eosin (H&E)-stained whole slide images (WSIs) of colon cancer patients into five distinct classes (tumor, stroma, necrosis, mucus, and background). The tumor–stroma ratio can be determined in the presence of necrotic or mucinous areas. We employ a few-shot model, eventually aiming for the easy adaptability of our approach to related segmentation tasks or other primaries, and compare the results to a well-established state-of-the art approach (U-Net). Both models achieve similar results with an overall accuracy of 86.5% and 86.7%, respectively, indicating that the adaptability does not lead to a significant decrease in accuracy. Moreover, we comprehensively compare with TSR estimates of human observers and examine in detail discrepancies and inter-rater reliability. Adding a second survey for segmentation quality on top of a first survey for TSR estimation, we found that TSR estimations of human observers are not as reliable a ground truth as previously thought.

In cancer, the activating transcription factor 2 (ATF2) has pleiotropic functions in cellular responses to growth stimuli, damage, or inflammation. Due to only limited studies, the significance of ATF2 in colorectal cancer (CRC) is not well understood. We report that low ATF2 levels correlated with worse prognosis and tumor aggressiveness in CRC patients. NanoString gene expression and ChIP analysis confirmed trophoblast cell surface antigen 2 (TROP2) as a novel inhibitory ATF2 target gene. This inverse correlation was further observed in primary human tumor tissues. Immunostainings revealed that high intratumoral heterogeneity for ATF2 and TROP2 expression was sustained also in liver metastasis. Mechanistically, our in vitro data of CRISPR/Cas9-generated ATF2 knockout (KO) clones revealed that high TROP2 levels were critical for cell de-adhesion and increased cell migration without triggering EMT. TROP2 was enriched in filopodia and displaced Paxillin from adherens junctions. In vivo imaging, micro-computer tomography, and immunostainings verified that an ATF2KO/TROP2high status triggered tumor invasiveness in in vivo mouse and chicken xenograft models. In silico analysis provided direct support that ATF2low/TROP2high expression status defined high-risk CRC patients. Finally, our data demonstrate that ATF2 acts as a tumor suppressor by inhibiting the cancer driver TROP2. Therapeutic TROP2 targeting might prevent particularly the first steps in metastasis, i.e., the de-adhesion and invasion of colon cancer cells.

The value of programmed cell death ligand-1 (PD-L1) to predict durable responses to immune checkpoint inhibitors (ICIs) in metastatic urothelial carcinoma (mUC) is inconsistent. We hypothesize that the use of archived primary tumor material (PRIM) for PD-L1 testing in clinical trials not properly reflecting the metastatic disease status (MET) contributes to this clinical issue.To analyze the predictive and prognostic value of PD-L1, spatial immunephenotypes, and major histocompatibility complex class I (MHC-I) determined in patient-matched PRIM/MET.PD-L1, spatial immunephenotypes, and MHC-I were examined in 154 mUC patients with at least one available pretreatment MET (138 patient-matched PRIM/MET pairs).PD-L1, spatial immunephenotype, and MHC-I status of (patient-matched PRIM and) pretreatment MET were correlated with chemotherapy and ICI response and outcomes.Discordance rates in patient-matched PRIM/MET were 25/30%, 36%, and 49% for PD-L1 (CPS10/IC5%), immunephenotypes, and MHC-I (loss vs preserved), respectively. Correlations with chemotherapy and ICI responses were observed for immunephenotypes and MHC-I status determined in MET (not for PD-L1 alone), but not in PRIM. In case of ICIs, patients with cytotoxic tumor immune microenvironment (TIME) showed durable responses with disease control rates of 90% and a hazard ratio for disease progression/death of 0.05 (95% confidence interval: 0.01-0.65) versus patients with immunedepleted MET (disease control rate 29%). MET MHC-I status added an incremental value to predict durable ICI responses. Limitations include the partly retrospective design and the lack of MET multisampling on individual patient level.The TIME is subject to substantial dynamics during metastatic evolution. MET immunephenotypes and MHC-I statuses show promising potential to predict chemotherapy and durable ICI responses, while the PRIM TIME does not. Thus, future clinical trials should rather rely on pretreatment MET biopsies reflecting the current immunological disease state than on PRIM.Prediction of chemotherapy and responses to immune checkpoint inhibitors might be possible using representative pretreatment metastatic biopsies.

Background: Antiangiogenic therapy is part of the guideline therapy of colorectal cancer (CRC). Surprisingly, the impact of the tumor microenvironment (TME) on tumor vessel endothelial cells (TECs) is largely unclear. The aim of this study was to investigate the presence of a TME-dependent transcriptional memory in TECs isolated from patients and to exploit it to retrieve signatures that characterize TECs in different intratumoral TMEs with impact on patient outcome. Methods: ECs from tumor and normal colon tissues, PBMCs and tumor cells were isolated from CRC patients with different prognostic TMEs. Cells were analyzed by qPCR, immunocytochemistry and multi-omics (transcriptomics, EPICmethylation chips, exome sequencing). Integrative bioinformatics was used to identify TME-dependent memory genes predicting prognosis and scRNASeq to validate endothelial gene expression in CRC tissues. Results: Ultrapure TECs were isolated from CRC with different prognostic TMEs (Th1 vs. non-Th1) and systematically compared by multi-omics. A transcriptional memory differentiating the respective TEC groups was identified. This in vivo imprinted transcriptional memory was preferentially regulated by epigenetic DNA methylation but not by genomic alterations and was different from an in vitro primed transcriptional memory to IFN-γ. Moreover, it was specific for TECs and not observed in CAFs. With integrative bioinformatics a TME-dependent memory signature was extracted and its expression in TECs in CRC tissues was confirmed by scRNASeq. Notably, the identified signature predicted the prognosis of CRC patients. Conclusion: We identified a tumor vessel-derived TME-dependent transcriptional memory signature that was manifested by epigenetic mechanisms and allowed tumor vessel-based prediction of CRC patients prognosis. Citation Format: Elisabeth Naschberger, Maximilian Fuchs, Nicholas Dickel, Meik Kunz, Charles G. Anchang, Richard Demmler, Bernt Popp, Arif B. Ekici, Steffen Uebe, Carol I. Geppert, Claudia Günther, Susanne Merkel, Vera S. Schellerer, Michael Stürzl. Vascular-derived signature imprinted by tumor microenvironment-dependent transcriptional memory predicts colon cancer prognosis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4587.

BackgroundLung cancer is the second common cancer type in western countries and has a high mortality. During the development and progression of the tumor, the nutrients in its environment play a central role. The tumor cells depend crucially on glucose metabolism and uptake. Tumor cell metabolism is dominated by the Warburg effect, where tumor cells produce large amounts of lactate from pyruvate under aerobic conditions. We thus reasoned that, reducing carbohydrates in the diet might support anti-tumoral effects of current immunotherapy and additionally target tumor immune escape.ObjectivesThe link between reducing carbohydrates to improve current immunotherapy is not clear. We thus aimed at analyzing the effects of different glucose levels on the tumor development, progression and the anti-tumoral immune response.MethodsWe correlated the clinical parameters of our LUAD cohort with different metabolic markers. Additionally, we performed cell culture experiments with A549 tumor cell line under different glucose levels. Lastly, we investigated the effect of low and high carbohydrate diet in an experimental murine model of lung cancer on the tumor progression and different immune subsets.ResultsHere we found a positive correlation between the body mass index (BMI), blood glucose levels, reduced overall survival (OS) and the expression of Insulin-like growth factor-1 receptor (IGF1R) in the lung tumoral region of patients with lung adenocarcinoma (LUAD). Furthermore, increasing extracellular glucose induced IGF1R expression in A549 LUAD cells. Functional studies in a murine model of LUAD demonstrated that, glucose restricted diet resulted in decreased tumor load in vivo. This finding was associated with increased presence of lung infiltrating cytotoxic CD8+ T effector memory (TEM), tissue resident memory T (TRM) and natural killer cells as well as reduced IGFR mRNA expression, suggesting that glucose restriction regulates lung immunity in the tumor microenvironment.ConclusionsThese results indicate that, glucose restricted diet improves lung immune responses of the host and suppresses tumor growth in experimental lung adenocarcinoma. As glucose levels in LUAD patients were negatively correlated to postoperative survival rates, glucose-restricted diet emerges as therapeutic avenue for patients with LUAD.

Purpose This single-centre cohort study was designed to identify factors that can predict primary tumour downstaging by neoadjuvant chemoradiotherapy (nCRT) in rectal carcinoma. Methods Prospectively collected data from 555 patients with clinical T category (cT) cT3-4 rectal carcinoma treated between 1995 and 2019 were retrospectively analysed. All patients received long-term neoadjuvant chemoradiotherapy followed by surgery with curative intent at the Department of Surgery, University Hospital Erlangen, Germany. Patient-, tumour- and treatment-related factors with a potential impact on the downstaging of rectal carcinoma to pathological T category (pT) ≤ ypT2 and ypT0 were analysed in univariate and multivariate logistic regression analyses. The prognosis of patients with and without downstaging of the primary tumour was compared. Results A total of 288 (51.9%) patients showed downstaging to ≤ ypT2. Eighty-six (15.5%) patients achieved clinical complete regression (ypT0). In the multivariate logistic regression analysis, the factors cT category, BMI, ECOG score, CEA, histological type, extension in the rectum and year of the start of treatment were found to be independent factors for predicting downstaging to ≤ ypT2 after neoadjuvant chemoradiotherapy. The year of treatment initiation also remained an independent significant predictor for pathological complete regression. The prognosis was superior in patients with downstaging to ≤ ypT2 in terms of locoregional and distant recurrence as well as disease-free and overall survival. Conclusion Factors predicting downstaging after long-term nCRT could be identified. This may be helpful for counselling patients and selecting the optimal treatment for patients with advanced rectal carcinoma.

Simple Summary The formation of metastasis is not only intricately orchestrated by cancer cells but is also affected by the surrounding extracellular matrix (ECM). The barrier function of the ECM represents an obstacle that cancer cells have to overcome to disseminate from the primary tumor to form metastasis in distant organs. Here, we demonstrate an approach to studying the remodeling of a collagen-rich ECM by colorectal tumor cells using multiphoton microscopy (MPM). This approach allows the analysis of the invasion front of tumors grown on the CAM in 3D. MPM is superior to conventional histology, which is limited to 2D analysis and needs extensive tissue preparation. Abstract Cancer cells facilitate tumor growth by creating favorable tumor micro-environments (TME), altering homeostasis and immune response in the extracellular matrix (ECM) of surrounding tissue. A potential factor that contributes to TME generation and ECM remodeling is the cytoskeleton-associated human death-associated protein kinase 1 (DAPK1). Increased tumor cell motility and de-adhesion (thus, promoting metastasis), as well as upregulated plasminogen-signaling, are shown when functionally analyzing the DAPK1 ko-related proteome. However, the systematic investigation of how tumor cells actively modulate the ECM at the tissue level is experimentally challenging since animal models do not allow direct experimental access while artificial in vitro scaffolds cannot simulate the entire complexity of tissue systems. Here, we used the chorioallantoic membrane (CAM) assay as a natural, collagen-rich tissue model in combination with all-optical experimental access by multiphoton microscopy (MPM) to study the ECM remodeling potential of colorectal tumor cells with and without DAPK1 in situ and even in vivo. This approach demonstrates the suitability of the CAM assay in combination with multiphoton microscopy for studying collagen remodeling during tumor growth. Our results indicate the high ECM remodeling potential of DAPK1 ko tumor cells at the tissue level and support our findings from proteomics.

Background The impact of body mass index (BMI) on prognosis in patients with curatively resected stage I–III colon carcinoma was analyzed. Methods The prospectively collected data of 694 patients who underwent complete mesocolic excision between 2003 and 2014 were analyzed. BMI was classified into four categories: underweight (BMI 2; n = 13), normal weight (BMI 18.5 to 24.9 kg/m2; n = 221), overweight (BMI 25.0 to 29.9 kg/m2; n = 309), and obese (BMI ≥ 30.0 kg/m2; n = 151). Univariate and multivariate analyses for comparison of prognosis were performed. Results The 5-year rate of locoregional recurrence in all 694 patients was 2.1%, and no differences were found with respect to BMI (p = 0.759). For distant metastasis, the 5-year rate for all patients was 13.4%, and BMI did not have a significant impact (p = 0.593). The 5-year rate of disease-free survival for all 694 patients was 72.4%. The differences with respect to BMI were not found to be significant in univariate analysis (p = 0.222). In multivariate Cox regression analysis, disease-free survival was significantly better in obese patients (HR 0.7; p = 0.034). Regarding overall survival, the 5-year rate for all patients was 78.1%. In univariate analyses, no significant differences were found for BMI (p = 0.094). In the Cox regression analysis, overweight and obese patients had significantly better survival (overweight: HR 0.7; p = 0.027; obese: HR 0.6; p = 0.019). Conclusion The better survival of overweight and obese patients in multivariate analyses must be interpreted with caution. It is influenced by several factors and seems to correspond to the phenomenon of the obesity paradox.

Neutrophil extracellular traps (NETs) are extracellular structures, composed of nuclear DNA and various proteins released from neutrophils. Evidence is growing that NETs exert manifold functions in infection, immunity and cancer. Recently, NETs have been detected in colorectal cancer (CRC) tissues, but their association with disease progression and putative functional impact on tumourigenesis remained elusive. Using high-resolution stimulated emission depletion (STED) microscopy, we showed that citrullinated histone H3 (H3cit) is sufficient to specifically detect citrullinated NETs in colon cancer tissues. Among other evidence, this was supported by the close association of H3cit with de-condensed extracellular DNA, the hallmark of NETs. Extracellular DNA was reliably differentiated from nuclear condensed DNA by staining with an anti-DNA antibody, providing a novel and valuable tool to detect NETs in formalin-fixed paraffin-embedded tissues. Using these markers, the clinical association of NETs was investigated in a cohort of 85 patients with colon cancer. NETs were frequently detected (37/85, 44%) in colon cancer tissue sections and preferentially localised either only in the tumour centre or both in the tumour centre and the invasive front. Of note, citrullinated NETs were significantly associated with high histopathological tumour grades and lymph node metastasis. In vitro, purified NETs induced filopodia formation and cell motility in CRC cell lines. This was associated with increased expression of mesenchymal marker mRNAs (vimentin [VIM], fibronectin [FN1]) and epithelial-mesenchymal transition promoting transcription factors (ZEB1, Slug [SNAI2]), as well as decreased expression of the epithelial markers E-cadherin (CDH1) and epithelial cell adhesion molecule (EPCAM). These findings indicated that NETs activate an epithelial-mesenchymal transition-like process in CRC cells and may contribute to the metastatic progression of CRC. © 2021 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

(1) Background: T-cell immunoglobulin and ITIM domain (TIGIT) is a potential immunotherapeutic target in a variety of malignant entities, and antibody-based treatments are currently under investigation in clinical trials. While promising results were observed in patients with lung cancer, the role of TIGIT in oral squamous cell carcinoma (OSCC) as a biomarker as well as a therapeutic target remains elusive. Therefore, we evaluated the role of TIGIT as a prognostic factor in OSCC. (2) Methods: Here, we describe the results of a retrospective tissue microarray (TMA) OSCC cohort. Using immunohistochemistry, TIGIT expression was correlated with overall and recurrence-free survival (OAS and RFS, respectively). Additionally, in silico analysis was performed based on the TCGA Head and Neck Squamous Cell Carcinoma (HNSCC) cohort in order to correlate patients’ survival with TIGIT and CD274 (encoding for PD-L1) gene expression levels. (3) Results: Database analysis revealed a beneficial outcome in OAS for tumor patients with high intraepithelial CD3-TIGIT-expression (n = 327). Hereby, OAS was 53.9 months vs. 30.1 months for patients with lower TIGIT gene expression levels (p = 0.033). In our retrospective OSCC-TMA cohort, elevated TIGIT levels on CD3+ cells correlated significantly with improved OAS (p = 0.025) as well as distant RFS (p = 0.026). (4) Conclusions: This study introduces TIGIT as a novel prognostic factor in OSCC, indicating the improved outcome of OSCC patients relative to their increased TIGIT expression. TIGIT might provide therapeutic implications for future immunotherapy in advanced-stage OSCC patients.

Purpose of the study: Tumor microenvironment (TME)-induced plasticity of fibroblasts and immune cells are known to contribute to cancer pathogenesis. In contrast, only little is known about the existence, stability, functions and prognostic potential of different plastic phenotypes of tumor vessel endothelial cells (TECs) in cancer. Recently, transcriptional memory effects have been observed in different cell lines after cytokine stimulation in vitro. From this we developed the hypothesis that isolated TEC may exhibit TME-dependent transcriptional memories, which may provide novel insights into the intratumoral effects on TEC plasticity in cancer. To investigate this, we isolated ultrapure TECs from colorectal cancer (CRC) with prognostically favorable (Th-1-like) or worse (non-Th1-like) immune-micro-environments and analyzed the prescence of TME-associated transcriptional memory patterns. Methods: TECs and as controls normal colon endothelial cells (NECs), PBMCs and tumor cells (isolated by laser-microdissection) from the same CRC patients (n=75) were isolated by FACS-sorting. Ultra high purity (98%) of TECs and NECs was validated by qPCR and immunocytochemistry. Quality validated complete sample sets from 12 patients (Th-1-like, n=6, non-Th1-like, n=6) were compared using exome sequencing, Clariome S chips and EPICmethylation. Integrative bioinformatics was used to identify differential gene sets and to predict their association with prognosis. Results: Differential transcriptional memory patterns were detected in TECs derived from CRC with different prognostic TMEs. Of note, the aggressiveness of the originating tumors was reflected by the functions of the vascular transcriptional memory genes. The transcriptional memory was imprinted by epigenetic DNA methylation and not due to acquired somatic variants in the genome. These results could be successfully confirmed by alternative methods. Most importantly, integrative bioinformatics analyses retrieved seven vascular memory genes (VMG), which were expressed in the TECs in a TME-dependent manner and predicted the outcome of patients in an independent cohort of CRC patients. Conclusion: Our findings demonstrate for the first time a TME-dependent transcriptional memory of TECs, providing a clinically relevant indicator of intra-tumoral TME-dependent vascular plasticity with perspectives for patient stratification in vessel-directed tumor therapies. Citation Format: Michael Stürzl, Maximilian Fuchs, Meik Kunz, Nicholas Dickel, Bernt Popp, Steffen Uebe, Arif B. Ekici, Carol I. Geppert, Susanne Merkel, Vera S. Schellerer, Elisabeth Naschberger. Micromilieu-dependent transcriptional memory of tumor vessel endothelial cells in colorectal cancer [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 3802.

The therapeutic index (TI) is a quantitative assessment of a drug safety proportional to its effectiveness. The estimation is intuitive when the engagement of the product with its target is dependent on stable chemistry and predictable pharmacokinetics as is the case for small molecules or antibodies. But for therapeutics with complex biodistribution and context-dependent potency such as adoptive cell therapy (ACT) products, TI estimations need to consider a broader array of factors. These include product-dependent variability such as functional fitness, unpredictable pharmacokinetics due to non-specific trapping, sequestration and extravasation into normal tissues and variable rates of in vivo expansion. In the case of solid malignancies, additional modifiers dependent on individual tumor immune biology may affect pharmacodynamics, including differential trafficking to benign compared with cancer tissue, hampered engagement with target cells, immune suppression and cellular dysfunction due to unfavorable metabolic conditions. Here, we propose a patient-specific assessment of factors affecting on-tumor from off-tumor activity in disparate immunologic environments that impact ACT’s clinical efficacy and may favorably balance the TI. for ACT products.

Immunotherapy improves the life expectancy of patients with lung cancer by targeting the immune inhibitory T cell surface markers resulting in the activation of anti-tumor T cell survival and suppression of tumor cell growth. The transcription factor repressor Positive Regulatory Domain Containing Protein 1 (encoded in humans by the Prdm1 gene) also known as B lymphocytes-induced maturation protein-1 (Blimp-1) affects the homeostasis and function of CD4+ and CD8+ T cells as well as T regulatory cells (T regs). We recently reported that Blimp-1 is induced in the lung tumoral region of patients with lung ADC. To further investigate whether Blimp-1 could be a candidate gene involved in the regulation of the anti-tumor immune-response in lung cancer, we started to investigate peripheral blood mononuclear cells (PBMCs) isolated from healthy control subjects and NSCLC patients for Blimp-1 expression in immunosuppressive CD4+CD25+Foxp3+ T regs. The PBMCs were cultured with and without antiCD3/CD28 antibodies, without and with TGF-beta, to mimic the tumor microenvironment, for 4 days and subsequently analyzed by flow cytometry using antibodies against Blimp-1, Foxp3, CD4 and CD25(IL-2R alpha chain). Here we found, by trend, induced CD4+Blimp1+CD25+Foxp3+ Tregs in unstimulated PBMCs from patients with lung cancer. Moreover, TGF-beta further induced these Blimp1+CD4+CD25+Foxp3+ Tregs in PBMCs obtained both from healthy controls and from NSCLC patients. Taken together these data indicate that the tumor microenvironment induces Blimp-1 in immunosuppressive CD4+CD25+Foxp3+ Tregs via TGF-beta. Thus, current immunotherapy should be combined to TGF-beta inhibitors to target CD4+CD25+Blimp1+Foxp-3+ T regulatory cells.

Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor activated by T-cell receptor (TCR) and Ca

Background: Nowadays, breast cancer represents both the most frequently diagnosed type of cancer as well as the leading cause of cancer associated death in women. Clinically, breast cancer tumors can be stratified into four major subgroups based on the expression of estrogen receptors (ER) and progesterone receptors (PR), the proliferation marker Ki67 and the expression/amplification of HER2/neu. Hormone receptor (HR) positive tumors generally have a slightly more favorable prognosis than the other two subgroups and can be treated with agents interfering with hormone signaling. A targeted therapy approach is also applied for HER2/neu amplified tumors by using agents that inhibit HER2/neu. In contrast, patients with triple negative breast cancers (TNBCs), i.e. tumors lacking HR expression as well as HER2/neu amplification, have a fairly poor outcome due to the high aggressiveness of this subgroup, especially if not responding to neoadjuvant therapy. Methods: In our study, we investigated phenotypical and functional characteristics of the two breast cancer cell lines MCF-7 and MDA-MB-231 that resemble the HR+ and the TNBC subgroup, respectively. In vitro, we were specifically interested in the invasiveness of the cells, thus determining their aggressiveness in a 3D spheroid invasion assay. In addition, we applied the in vivo chorioallantoic membrane (CAM) xenograft assay to histologically analyze the tumor growth patterns and evaluate metastasis formation using a human-specific Alu-PCR method and an in vivo imaging system (IVIS). Results: As anticipated, the highly aggressive TNBC cell line MDA-MB-231 generated CAM micro-tumors much larger than those derived of the HR+ MCF-7 cell line. Moreover, we were able to verify different characteristic histological features of TNBC and HR+ tumors in the CAM xenograft model. In this regard, the MDA-MB-231 cells showed the distinct growth pattern of a poorly differentiated adenocarcinoma and a highly infiltrative growth into the CAM with strong tumor budding (clusters of ≤5 cells) at the invasive front. The high tumor budding rate could be correlated to an extremely invasive potential of the TNBC cells in vitro as well as to a strong metastasis formation in the in vivo CAM assay. Conclusion: Our findings suggest that the CAM xenograft assay represents a suitable model to mimic the clinical situation of breast cancer patients. For the first time, we could show that tumor budding is correlated with metastasis formation in this alternative xenograft system. Citation Format: Julienne K. Muenzner, Raphela A. Ranjan, Markus Eckstein, Ramona Erber, Philipp Kunze, Carol I. Geppert, Matthias Ruebner, Tobias Baeuerle, Arndt Hartmann, Regine Schneider-Stock. Tumor budding: A predictor for metastasis in the CAM xenograft model [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 3699.

Preventing ischemia-related cell damage is a priority when preserving tissue for transplantation. Perfusion protocols have been established for a variety of applications and proven to be superior to procedures used in clinical routine. Extracorporeal perfusion of muscle tissue though cumbersome is highly desirable since it is highly susceptible to ischemia-related damage. To show the efficacy of different perfusion protocols external field stimulation can be used to immediately visualize improvement or deterioration of the tissue during active and running perfusion protocols. This method has been used to show the superiority of extracorporeal perfusion using porcine rectus abdominis muscles perfused with heparinized saline solution. Perfused muscles showed statistically significant higher ability to exert force compared to nonperfused ones. These findings can be confirmed using Annexin V as marker for cell damage, perfusion of muscle tissue limits damage significantly compared to nonperfused tissue. The combination of extracorporeal perfusion and external field stimulation may improve organ conservation research.

We used high-resolution mass spectrometry to measure the abundance of more than 9,000 proteins in 19 individually dissected colorectal tumors representing lymph node metastatic (n = 10) and nonmetastatic (n = 9) phenotypes. Statistical analysis identified MX1 and several other proteins as overexpressed in lymph node-positive tumors. MX1, IGF1-R and IRF2BP1 showed significantly different expression in immunohistochemical validation (Wilcoxon test p = 0.007 for IGF1-R, p = 0.04 for IRF2BP1 and p = 0.02 for MX1 at the invasion front) in the validation cohort. Knockout of MX1 by siRNA in cell cultures and wound healing assays provided additional evidence for the involvement of this protein in tumor invasion. The collection of identified and quantified proteins to our knowledge is the largest tumor proteome dataset available at the present. The identified proteins can give insights into the mechanisms of lymphatic metastasis in colorectal carcinoma and may act as prognostic markers and therapeutic targets after further prospective validation.