Supplementary Figure Legends 1-4 from Variable Inhibition of Thrombospondin 1 against Liver and Lung Metastases through Differential Activation of Metalloproteinase ADAMTS1

PDF file - 360KB, aCGH analysis of chromosomal aberrations in single CRC-derived CTC and corresponding tumor tissue (S1); An alternative aCGH algorithm confirms the tumor cell identity of the examined single CTC (S2); Validation of the reproducibility of low cell number real-time PCR (S3); Establishment and performance of single cell realtime PCR analysis (S4); Picture of a cluster of 3 CTC from patient HD-2095 (S5).

PDF file - 55K, Primer sequences used for qRT-PCR.

Supplementary Table S4. GSEA results metastatic signature.

Supplementary Table S1. Disease microarrays GEO accession.

Supplementary Data from A Complex of EpCAM, Claudin-7, CD44 Variant Isoforms, and Tetraspanins Promotes Colorectal Cancer Progression

PDF file - 96KB, Summary of patient characteristics and number of chromosomal aberrations determined by aCGH analyses (S1); Summary of the full genomic analyses of CRC derived CTC and the corresponding tumor tissue (S2); UICC stages of the patients enrolled in the mutational analysis (S3); Characteristics of the patients enrolled in the CTC transcriptional analysis (S4); Distribution of sex and UICC stage in patients enrolled in the CTC transcriptional analysis (S5); Summary of patient characteristics and source of CTC isolation for the CTC transcriptional analysis (S6); Clinical information regarding patients enrolled in the CTC mutational analysis (S7); Overview of the real-time PCR primer sequences used in the CTC transcriptional analysis (S8); Overview of the pre-amplification PCR primer sequences used for the mutational profiling of patient-derived CTC and tumor tissue (S9); Summary of the primer sequences used for sequencing analyses and detection of common CRC mutation (S10); Summary of the primer sequences used for noncoding and coding MSI analyses (S11).

Supplementary Figures S1-S5; Supplementary Table S1.

Supplementary Figure S1. Benchmarking anti-correlated drug-disease relations with known drug indications. Supplementary Figure S2. Genes in the metastatic gene profile. Supplementary Figure S3. Fraction of apoptotic cells after treatment with candidate drugs. Supplementary Figure S4. Four circulating tumor cells.

Supplementary Table S2. Drug-disease relations.

Supplementary Figure 2 from Variable Inhibition of Thrombospondin 1 against Liver and Lung Metastases through Differential Activation of Metalloproteinase ADAMTS1

Although tumor‐initiating cell ( TIC ) self‐renewal has been postulated to be essential in progression and metastasis formation of human pancreatic adenocarcinoma ( PDAC ), clonal dynamics of TIC s within PDAC tumors are yet unknown. Here, we show that long‐term progression of PDAC in serial xenotransplantation is driven by a succession of transiently active TIC s producing tumor cells in temporally restricted bursts. Clonal tracking of individual, genetically marked TIC s revealed that individual tumors are generated by distinct sets of TIC s with very little overlap between subsequent xenograft generations. An unexpected functional and phenotypic plasticity of pancreatic TIC s in vivo underlies the recruitment of inactive TIC clones in serial xenografts. The observed clonal succession of TIC activity in serial xenotransplantation is in stark contrast to the continuous activity of limited numbers of self‐renewing TIC s within a fixed cellular hierarchy observed in other epithelial cancers and emphasizes the need to target TIC activation, rather than a fixed TIC population, in PDAC .

The immune response influences the clinical course of colorectal cancer (CRC). Analyzing the invasive margin of human CRC liver metastases, we identified a mechanism of immune cell exploitation by tumor cells. While two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10, CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5. CCR5 blockade in patient-derived functional in vitro organotypic culture models showed a macrophage repolarization with anti-tumoral effects. These anti-tumoral effects were then confirmed in a phase I trial with a CCR5 antagonist in patients with liver metastases of advanced refractory CRC. Mitigation of tumor-promoting inflammation within the tumor tissue and objective tumor responses in CRC were observed.

Abstract Background: Hepatocellular carcinoma (HCC) is the 3rd most leading cause of cancer death in the world. Despite extensive research, HCC has a strikingly bad prognosis due to the limited therapeutic options: Other than surgical resection, there is no curative treatment. No cytotoxic agent has shown efficacy so far, and the only currently approved systemic agent, sorafenib, is only used in a palliative context. This dilemma is due to the therapy-resistant biology as well as its variable pathogenesis and biological diversity, which requires excellent preclinical models to allow differential and innovative studies. Innovative 3D culture methods such as organoid culture have been increasingly implemented in routine in vitro research for many tumor entities. However, due to its distinct biology, organoid culture of HCC has been a challenge. We here describe the initial establishment and characterization of HCC organoid culture. Materials/Methods: Surgical HCC tissue specimens were obtained in the operating rooms of the University Hospital Dresden; biopsy specimens were obtained via CT-/ultrasound-guided biopsy of HCC lesions. All samples were processed according to a novel protocol and cultured in Matrigel supplemented with a highly specialized culture medium. Organoids were propagated, passaged and frozen at regular intervals. Proliferation was measured using a modified WST proliferation assay protocol. Next-generation sequencing (NGS) was performed on corresponding triplets of healthy tissue, tumor tissue and organoid samples using the TruSight One Sequencing Panel (Illumina) on an Illumina MiSeq Sequencer. Histology was performed on formalin-fixed and paraffin-embedded organoids using standard protocols. Results: We were able to establish a method to culture HCC samples as organoids in vitro. Proliferation assays revealed distinct differences in organoid growth of HCCs from different patients and correlated well with both clinical behavior and histological grading of the tumors. Amplicon sequencing (targeting 4,813 cancer-associated genes) of corresponding sets of tumor specimens and HCC organoids demonstrated the HCC origin of the organoids and their genetic and genomic stability in culture. Treatment of HCC organoids with sorafenib showed the expected reduction in proliferation and viability. Interestingly, treatment of the HCC organoids with hepatocyte growth factor (HGF) resulted in a marked reduction in proliferation as opposed to healthy liver organoids, which had increased proliferation activity upon stimulation with HGF. Histology of the HCC organoids showed distinct features of HCC and corresponded well with the corresponding HCC tissue samples. Conclusion: We successfully established a protocol reliably allowing 3D organoid culture of HCC. The organoids faithfully recapitulate the biology of the corresponding tumor and can be used for multiple purposes including in vitro therapeutic testing. Citation Format: Sebastian Schölch, Lahiri K. Nanduri, Daniel Kühn, Jürgen Weitz, Moritz Koch, Nuh N. Rahbari. Establishment and initial characterization of human hepatocellular carcinoma organoid culture. [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 4263.

BACKGROUND:Matrixmetalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases which are involved in angiogenesis, tumor invasion and metastatic formation. Up to date, the prognostic relevance of MMPs in serum of patients with colon cancer remains unknown. Thus, we wanted to assess an expression pattern of MMPs in a homogenous cohort of colon cancer patients to assess their potential as prognostic biomarkers.METHODS:Differences in the expression pattern of MMP7, MMP10 and MMP12 in 78 serum specimens of patients with an adenocarcinoma of the colon and serum specimens of a healthy control group were assessed using Luminex-100 technologies. Subsequently, we correlated these results with histopathological and clinical data of the patients.RESULTS:Luminex based expression analysis revealed a significant overexpression of MMP7 and an overexpression of MMP10 and MMP12 in the sera of colon cancer patients compared to the healthy control group. Patients with vascular invasion showed a significantly higher MMP12 expression than V0-staged patients. Moreover overexpression of MMP7, MMP10 and MMP12 in colon cancer patients´ sera displayed a significantly impaired overall survival. Multivariate analysis revealed high MMP10 serum levels to be an independent adverse prognostic marker in colon cancer patients.CONCLUSIONS:Expression patterns of MMP7, MMP10 and MMP12 in colon cancer patients´ sera are different compared to serum specimens of healthy individuals. Furthermore, overexpression of MMP7, MMP10 and MMP12 in colon cancer patients´ sera correlates with a dismal prognosis and may help to stratify patients into different risk groups.

BACKGROUND:Signal transducer and activator of transcription proteins (STATs) are crucial regulators of cell growth and differentiation; however, their specific prognostic impact in human colon cancer has only been studied to limited extent. We aimed to assess the prognostic significance of specific STAT expression patterns in colon carcinoma.METHODS:Protein expression patterns of activated STAT1, STAT3, STAT4, and STAT5 in human colon carcinoma tissue and corresponding healthy mucosa (n = 104) were assessed using multiplex bead-based immunoassay technologies. Expression patterns were correlated with clinical and survival data. Immunohistochemistry was performed to assess spatial expression of STAT3 and STAT5.RESULTS:STAT3 was underexpressed whereas STAT4 and STAT5 were overexpressed in colon carcinoma tissue. Primary tumors from patients with distant metastases (M1) displayed significantly increased expression of STAT1 and STAT3 but decreased expression of STAT4 and STAT5. Increased tumor expression of STAT1 or STAT3 was associated with impaired patient survival, whereas increased expression of STAT4 or STAT5 correlated with improved survival. Multivariate analysis identified an increased STAT3/STAT5 expressional ratio as an adverse prognostic marker in colon cancer patients.CONCLUSIONS:The tumor progression-associated transcription factors STAT3, STAT4, and STAT5 are differently expressed in colon carcinoma tissue and colon mucosa. Moreover, the STAT3/STAT5 expression ratio is an independent prognostic marker in colon cancer patients.

The composition of tumor-targeted T cell infiltrates is a major prognostic factor in colorectal cancer (CRC) outcome; however, the functional role of these populations in prolonging patient survival remains unclear. Here, we evaluated 190 patients with CRC for the presence of functionally active tumor-infiltrating lymphocytes (TILs), the tumor specificity of these TILs, and the correlation between patient TILs and long-term survival. Using intracytoplasmic cytokine staining in conjunction with HLA multimers loaded with tumor peptide and antigen-specific cytokine secretion assays, we determined that TNF-α expression delineates a population of tumor antigen-specific (TA-specific) cytotoxic T lymphocytes (CTLs) present within tumors from patients with CRC. Upregulation of TNF-α expression in TILs strongly correlated with an increase in the total amount of intratumoral TNF-α, which is indicative of tumor-specific CTL activity. Moreover, a retrospective multivariate analysis of 102 patients with CRC, which had multiple immune parameters evaluated, revealed that increased TNF-α concentration was an independent prognostic factor. Together, these results indicate that the prognostic impact of T cell infiltrates for CRC maybe largely based on subpopulations of active TA-specific T cells within the tumor, suggesting causal implication for these cells in patient survival. Additionally, these results support the use of intratumoral TNF-α, which is indicative of T cell function, as a prognostic parameter for CRC.

Background Radical resection is the treatment of choice for colorectal liver metastases (CLM). Unfortunately, only about 20 % of patients present with initially resectable disease, in most cases due to bilobar disease. In the last two decades, major achievements have been made to extend surgical indications to patients with bilobar CLM, such as two-stage hepatectomy with or without portal vein occlusion and associating liver partition and portal vein ligation for staged hepatectomy (ALPPS). Purpose The purpose of this review article was to summarize current surgical approaches and their safety and efficacy for patients with initially unresectable bilobar CLM. Conclusion In selected patients, two-stage hepatectomy and ALPPS are efficient and safe to convert unresectable to resectable CLM. Further studies are required to evaluate long-term outcome of these procedures.

In addition to local cytotoxic activity, radiotherapy may also elicit local and systemic antitumor immunity, which may be augmented by immunotherapeutic agents including Toll-like receptor (TLR) 7/8 agonists. Here, we investigated the ability of 3M-011 (854A), a TLR7/8 agonist, to boost the antigen-presenting activity of dendritic cells (DC) as an adjuvant to radiotherapy. The combined treatment induced marked local and systemic responses in subcutaneous and orthotopic mouse models of colorectal and pancreatic cancer. In vitro cytotoxicity assays as well as in vivo depletion experiments with monoclonal antibodies identified NK and CD8 T cells as the cell populations mediating the cytotoxic effects of the treatment, while in vivo depletion of CD11c(+) dendritic cells (DC) in CD11c-DTR transgenic mice revealed DC as the pivotal immune hub in this setting. The specificity of the immune reaction was confirmed by ELISPOT assays. TLR7/8 agonists therefore seem to be potent adjuvants to radiotherapy, inducing strong local and profound systemic immune responses to tumor antigens released by conventional therapy.

Epithelial‐to‐mesenchymal transition (EMT) contributes significantly to tumor progression and metastasis. The assessment of EMT‐associated transcription factors could be a promising approach to identify biomarkers and potential therapeutic targets in colorectal cancer. In our study, we focused on the transcription factor “Sine oculis homeobox” (SIX) 1, which is a member of the superfamily of the homeobox genes and has been described to promote EMT in different types of tumors. Immunohistochemistry against SIX1 was performed on colorectal mucosa, adenomas, carcinomas‐in situ and primary adenocarcinomas. An expression score was developed and subsequently assessed for its prognostic value in two independent cohorts. Cohort 1 consisted of 128 patients with stage I–III colorectal cancer; cohort 2 included 817 patients with stage I–III colorectal cancer who had participated in the DACHS study. HCT‐116 cells were transfected with SIX1 plasmids and subjected to migration and colony formation assays. The expression of SIX1 increases gradually from mucosa to colorectal adenocarcinomas (p > 0.0001). Univariate and multivariate analyses reveal that high expression of SIX1 is associated with decreased overall survival (cohort 1: HR: 4.01, CI: 1.20–14.07, p = 0.025; cohort 2: HR: 1.43, CI: 1.014–2.02, p = 0.047). Overexpression of SIX1 induces a more mesenchymal‐like phenotype in HCT‐116 cells and enhances tumor migration. High expression of SIX1 is an independent prognostic marker in colorectal cancer. It might be a promising biomarker to stratify patients into different risk groups. Moreover, targeting SIX1 might be a novel therapeutic approach in patients with colorectal cancer.