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Background: Tumour immunoprofiling captures tumour-microenvironment interactions and enables precision medicine. Multiplex immunofluorescence (mIF) imaging can provide comprehensive quantitative and spatial information for multiple immune markers. However, application at scale to clinical trial samples sourced from multiple institutions is challenging due to pre-analytical heterogeneity. Methods: We analysed 12'592 tissue microarray (TMA) spots from 3'545 colorectal cancers (CRC) sourced from more than 240 institutions in two clinical trials (QUASAR 2 and SCOT) stained for CD4, CD8, CD20, CD68, FoxP3, pan-cytokeratin and DAPI by mIF. TMA cores were multi-spectrally imaged by digital pathology and analysed by cell-based and pixel-based marker analysis. We developed and validated an adaptive thresholding method to account for inter- and intra-slide intensity variation in TMA analysis, compared the numerical results between analysis approaches and validated against the current gold standard of single-plex chromogenic immunohistochemistry (IHC). Results: Adaptive thresholding at a slide and spot level effectively ameliorated inter- and intra-slide intensity variation enabling high-throughput analysis of mIF-stained TMA cores by digital pathology and improving the image analysis results compared to methods using a single global threshold. Comparison of our mIF approach with CD8 IHC data derived from subsequent sections indicates the validity of our method (Spearman's rank correlation coefficients (SCC) between 0.63 and 0.66, p-value << 0.01). Evaluation of correlation between cell-based and pixel-based analysis results confirms equivalency (SCC > 0.8, p<< 0.01, except for CD20 in epithelium region) of both analytical approaches for precision immunoprofiling by mIF. Conclusions: This study reports an analytical approach to the largest multiparameter immunoprofiling study of clinical trial samples to date and establishes an adaptive thresholding method to account for inter- and intra-slide variation introduced by pre-analytical heterogeneity. This approach can enable the application of mIF immunoprofiling of clinical trial TMA datasets at scale.

Separate depiction of immunohistochemical staining of p16INK4a in situ.

List of transgenic mice used in the study.

Supplementary Figure legends

Reduction of β-catenin (CTNNB1) destroying complex components, for example, adenomatous polyposis coli (APC), induces β-catenin signaling and subsequently triggers activation of genes involved in proliferation and tumorigenesis. Though diminished expression of APC has organ-specific and threshold-dependent influence on the development of liver tumors in mice, the molecular basis is poorly understood. Therefore, a detailed investigation was conducted to determine the underlying mechanism in the development of liver tumors under reduced APC levels. Mouse liver at different developmental stages was analyzed in terms of β-catenin target genes including Cyp2e1, Glul, and Ihh using real-time RT-PCR, reporter gene assays, and immunohistologic methods with consideration of liver zonation. Data from human livers with mutations in APC derived from patients with familial adenomatous polyposis (FAP) were also included. Hepatocyte senescence was investigated by determining p16INK4a expression level, presence of senescence-associated β-galactosidase activity, and assessing ploidy. A β-catenin activation of hepatocytes does not always result in β-catenin positive but unexpectedly also in mixed and β-catenin–negative tumors. In summary, a senescence-inducing program was found in hepatocytes with increased β-catenin levels and a positive selection of hepatocytes lacking p16INK4a, by epigenetic silencing, drives the development of liver tumors in mice with reduced APC expression (Apc580S mice). The lack of p16INK4a was also detected in liver tumors of mice with triggers other than APC reduction.Implications: Epigenetic silencing of p16Ink4a in selected liver cells bypassing senescence is a general principle for development of liver tumors with β-catenin involvement in mice independent of the initial stimulus. Mol Cancer Res; 13(2); 239–49. ©2014 AACR.

This file contains Supplementary Materials and Methods, Supplementary Figures, Legends to Supplementary Figures and Supplementary Tables. Figure S1 shows histopathological analysis of Ptenfl/fl and Ptenfl/fl Erk5fl/fl tumours at 3 and 9-10 month fixed time points and demonstrates that there is reduced %Ki67 positive nuclei in Ptenfl/fl Erk5fl/fl tumours at the 9-10 month time point. There is very little T lymphocyte infiltration at the 3 month time point but significant T lymphocyte infiltration is observed at the later time point. Figure S2 shows Western Blot and QPCR data to validate that P1 and PE cells are null for PTEN and ERK5 protein and Erk5 mRNA transcript where expected. Also shown is IHC staining demonstrating increased immunoreactivity for CCL5 and CXCL10 in Ptenfl/fl Erk5fl/fl compared to Ptenfl/fl tumours and that CCR5 and CXCR3 immunoreactivity is detected in the tumour epithelium and immune cells present in the stroma of Ptenfl/fl Erk5fl/fl tumours. Figure S3 shows IHC staining for CD45 demonstrating significant lymphocyte infiltration in Ptenfl/fl and Ptenfl/fl Erk5fl/fl tumours. Also shown is dual IHC staining for CD3/CD4 revealing a high degree of CD4 reactivity within the lymphoid aggregates of Ptenfl/fl Erk5fl/fl tumours.

Prostate cancer does not appear to respond to immune checkpoint therapies where T-cell infiltration may be a key limiting factor. Here, we report evidence that ablating the growth regulatory kinase Erk5 can increase T-cell infiltration in an established Pten-deficient mouse model of human prostate cancer. Mice that were doubly mutant in prostate tissue for Pten and Erk5 (prostate DKO) exhibited a markedly increased median survival with reduced tumor size and proliferation compared with control Pten-mutant mice, the latter of which exhibited increased Erk5 mRNA expression. A comparative transcriptomic analysis revealed upregulation in prostate DKO mice of the chemokines Ccl5 and Cxcl10, two potent chemoattractants for T lymphocytes. Consistent with this effect, we observed a relative increase in a predominantly CD4+ T-cell infiltrate in the prostate epithelial and stroma of tumors from DKO mice. Collectively, our results offer a preclinical proof of concept for ERK5 as a target to enhance T-cell infiltrates in prostate cancer, with possible implications for leveraging immune therapy in this disease. Cancer Res; 77(12); 3158–68. ©2017 AACR.

Free Article from Systems Biologists Seek Fuller Integration of Systems Biology Approaches in New Cancer Research Programs

Systems biology takes an interdisciplinary approach to the systematic study of complex interactions in biological systems. This approach seeks to decipher the emergent behaviors of complex systems rather than focusing only on their constituent properties. As an increasing number of examples illustrate the value of systems biology approaches to understand the initiation, progression, and treatment of cancer, systems biologists from across Europe and the United States hope for changes in the way their field is currently perceived among cancer researchers. In a recent EU-US workshop, supported by the European Commission, the German Federal Ministry for Education and Research, and the National Cancer Institute of the NIH, the participants discussed the strengths, weaknesses, hurdles, and opportunities in cancer systems biology. Cancer Res; 70(1); 12–3

Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterised by fetal transcriptional profiles. Neoplasia hijacks regenerative signalling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signalling disruption, and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis, and is influenced by cell-intrinsic, extrinsic and therapeutic selective pressures.

196 Background: The ESMO clinical practice guidelines recommend consideration of Immunoscore (IS) for risk assessment of early colon cancer patients. IS clinical performance was assessed in the SCOT and IDEA-HORG trials evaluating 3 vs. 6 months (3m vs. 6m) of mFOLFOX6 adjuvant chemotherapy in stage III colorectal cancer (CRC). Methods: 1,002 formalin-fixed paraffin-embedded (FFPE) tumor samples (762 from SCOT;240 from HORG) were collected, of which 851 were eligible for biomarker analysis. Eligible samples were classified into 2 groups using pre-defined cut-offs (IS-Low, IS- High) and the performance of IS to predict 3 year disease-free survival (3y-DFS) was evaluated. Results: IS was successfully assessed in 846 cases (99%). 615 (72.7%) samples were classified as IS-High (311 and 304 in 3m and 6m arm, respectively). No significant association between IS and patients’ gender, age, PS, BMI or primary tumour location was observed. However, a significant difference between IS-High (43.7%) and IS Low (57.1%) was observed in the proportion of high risk (T4 and/or N2) tumours (p=0.001). Patients with IS-High tumors had significantly longer 3y-DFS (79.4%, 95%CI: 75.9%-82.4%) compared to those with IS-Low tumors (65.0%, 95%CI: 58.3%-70.9%); adjusted hazard ratio (HR) 1.9 (95%CI: 1.46-2.46; p

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes., Kras is often mutated in colorectal cancer but how this oncogenic mutation alters signalling pathways globally is undetermined. Here, the authors analyse how this mutation affects protein interaction networks and signal flow showing an extensive re‐wiring of signalling in response to KRas mutation

Colorectal cancers (CRCs) most commonly develop from adenomas harboring a mutant APC gene that leads to aberrant activation of the canonical Wnt/ß-catenin pathway. One of the mechanisms of adenoma progression involves the PROX1 transcription factor that promotes tumor dysplasia and progression in CRC. In the normal intestinal epithelial cells, ligands of the R-Spondin (RSPO) family are able to enhance the canonical Wnt pathway activity by binding to receptor proteins of the LGR family, such as LGR5. Tumorigenic RSPO gene fusions have recently been discovered in CRCs, but the role of RSPOs in cancer is incompletely known. We previously reported that TGFß induced apoptosis of APC-mutant organoids, including the LGR5+ stem cells, was mediated by the proapoptotic protein BIM, whereas wild-type intestinal crypts were markedly less sensitive to TGFß. The KRAS oncogene increased apoptosis resistance in the adenomas via activation of the Erk1/2 kinase pathway, which led to Bim down-regulation and increased resistance to TGFß. A subsequent study showed that RSPO/LGR5 can inhibit the in vitro growth of cultured human CRC cell line by activating the TGFß/SMAD signaling pathway. Because we were interested in analyzing the effect of exogenous Wnt signals on PROX1 expression in intestinal tumorigenesis, we constructed AAV-vectors for systemic expression of a soluble RSPO1 protein in ApcMin/+ mice. We found that the RSPO1-Fc fusion protein suppresses the Wnt/ß-catenin signaling activity in intestinal adenomas and in adenoma-derived intestinal organoids ex vivo, but not in normal intestinal epithelial cells. In the Apc mutant cells, the RSPO1-Fc fusion protein activated the TGFß/SMAD signaling pathway to suppress several Wnt target genes and adenoma growth, which effect was rescued suppressed by the TGFß receptor kinase inhibitor SB-431542. Simultaneously, RSPO1-Fc induced proliferation of the normal intestinal stem cells, giving them a growth advantage over the mutant cells, which enabled the intestinal epithelium to eventually outgrow the adenoma cells. Prolonged systemic expression of AAV-RSPO1-Fc decreased significantly the number of the intestinal adenomas and improved the overall survival of ApcMin/+ mice. Thus RSPO1-Fc provides the normal intestinal epithelial cells a growth advantage when compared to the adenoma cells, which eventually leads to the extrusion of the adenomatous tissue. An attractive idea now is to exploit such differential response of normal vs. cancer cells in cancer therapy. Citation Format: Marianne Lähde, Sarika Heino, Jenny Högström, Veli-Matti Leppänen, Seppo Kaijalainen, Olli Ritvos, Owen Sansom, Kari Alitalo. Systemic RSPO1 delivery leads to regression of intestinal adenomas via activation of the TGFβ/SMAD pathway [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 LB-094.

Prostate cancer (PCa) does not appear to respond to immune checkpoint therapies where T cell infiltration may be a key limiting factor. Here we report evidence that ablating the growth regulatory kinase Erk5 can increase T cell infiltration in an established Pten-deficient mouse model of human PCa. Mice that were doubly mutant in prostate tissue for Pten and Erk5 (prostate DKO) exhibited a markedly increased median survival with reduced tumor size and proliferation compared to control Pten-mutant mice, the latter of which exhibited increased Erk5 mRNA expression. A comparative transcriptomic analysis revealed upregulation in prostate DKO mice of the chemokines Ccl5 and Cxcl10, two potent chemoattractants for T lymphocytes. Consistent with this effect, we observed a relative increase in a predominantly CD4+ T cell infiltrate in the prostate epithelial and stroma of tumors from DKO mice. Collectively, our results offer a preclinical proof of concept for ERK5 as a target to enhance T cell infiltrates in prostate cancer, with possible implications for leveraging immune therapy in this disease.

One question that has been central to the study of the Apc gene is why the Apc gene is mutated so frequently in colorectal cancer but relatively infrequently in other tumour types. This chapter reviews recent data obtained in mice after conditional deletion of both copies of the Apc gene from adult epithelial tissues with particular focus on the intestinal epithelium. These data suggest that a major reason for the frequent mutation of Apc in colorectal cancer lies in the distinct character of the intestinal epithelium where Apc loss leads to a progenitor-like phenotype. Thus intestinal enterocytes lacking Apc escape the two major selective constraints that usually prevent cells from becoming cancerous in the intestine: they fail to differentiate and fail to migrate so are not sloughed off into the intestinal lumen.

The mammalian intestinal epithelium is one of the most actively self-renewing tissues, which is constantly replenished by pluripotent intestinal stem cells (ISCs). This remarkable characteristic seems to impact in its high propensity for malignant transformation. Indeed, many of the molecular pathways that regulate normal intestinal homeostasis appear involved in colorectal carcinogenesis. Inactivating mutations of the APC (Adenomatous Polyposis Coli) gene is a hallmark of colorectal cancer. The main tumor suppressive function of Apc is to negatively regulate Wnt signaling. Targeted deletion of Apc in the murine intestine, and more recently in the zebrafish gut, recapitulate many aspects of the human disease. Work in Drosophila now reveals that the role of APC in the intestine is ancient and highly conserved across species. In support of these findings, we present data which suggests that APC1 may be a marker for adult ISCs in Drosophila and is required specifically within the ISCs to regulate intestinal homeostasis. Here we discuss the similarities and differences between these model organisms in regards to the role of Wnt signaling and APC in intestinal homeostasis and transformation.