Early Tumor Development Research Articles

Abstract 4966: Characteristics of immune repertoires in early lung cancer patients

Abstract Lung cancer is the leading cause of cancer death in the world, and early lung cancer detection is vital for improving the overall survival. Besides x-ray and CT scan, few genetic tests have been widely used for detecting early lung tumors. We hypothesize that early lung cancer patients have some unique features in the immune repertoires which can be utilized for early cancer detection and profiling disease development. In this study, we performed RNA-seq for paired tumor and tumor-adjacent normal samples from 59 early lung adenocarcinoma patients. We found that early lung tumors have more cytokine activities, higher B cell and CD4+ T cell infiltration, and lower CD8+ T cell infiltration compared to normal lung tissues. We assembled B cell receptors from RNA-seq reads and found more B cell clonal expansion and somatic hypermutations in the tumor than in tumor-adjacent normal tissues, similar to the B cell repertoires in late-stage lung cancer patients. In both early- and late-stage lung tumors, we identified a few cytokines that positively correlated with more IgA isotypes and associated with patient survival. To gain more insights on the B cell binding specificity, we performed motif enrichment analysis for paired tumor and normal samples and validated some tumor-enriched receptor motifs in matched patient blood samples, which implies a new way of early cancer detection. More interesting, we assembled microbial transcripts from RNA-seq data and identified some known lung disease-related species in both tumor and normal samples. The complex interactions between cancer cells, B cells, and microbiota in the early lung tumor development are waiting to be elucidated. Citation Format: Jin Wang, Xihao Hu, Peng Zhang, Xiaole Shirley Liu. Characteristics of immune repertoires in early lung cancer patients [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 4966.

Abstract 5149: Subclonal sociability: Interactions of HER2 and PIK3CAmutant cells in breast cancer

Abstract Background: Tumor heterogeneity is a common characteristic of many cancers and plays an important role in their development and eventual metastasis. The traditional clonal evolution model describes cancer cells as descendants of a single progenitor that has accumulated a stepwise series of mutations. However, recent sequencing studies have revealed the presence of genetically distinct subclones in numerous tumors. PIK3CA is the most commonly mutated gene in breast cancer while HER2 is overexpressed in ~20% of breast cancers. Activating missense mutations in the gene that encodes HER2 have also been discovered, and our lab has previously shown the effects of these mutations and their synergy with PIK3CA mutations in a double-mutant cell. This project explores the interactions between cells with either a HER2 or a PIK3CA mutation when they are present as genetically distinct subclones in co-cultures. Methods: Isogenic MCF-10A cells containing mutations in PIK3CA, HER2, or no mutation were co-cultured in growth factor-deprived conditions. In these conditions, PIK3CA mutant cells are known to be able to grow, while HER2 mutant and wild-type (WT) cells arrest. The cellular makeup of the co-cultures was determined using droplet digital PCR (ddPCR) with probes against the respective mutations. Cells were tagged with stable expression of either zsGreen (PIK3CA mutants) or tdTomato (HER2 and WT) and observed under fluorescent microscopy. Further imaging was performed using the IncuCyte live cell imaging system (Essen Bioscience), which allowed for periodic imaging of cells in culture. Co-cultures were treated with Neratinib or Alpelisib (BYL719), which are HER1/2/4 and PIK3CA inhibitors, respectively. Cells in each condition were counted and ddPCR was performed to determine allelic fractions of the co-cultures. Results: When cultured together, PIK3CA mutant cells can induce proliferation in HER2 mutant cells, but not WT cells. Over time, HER2 mutant cells grow to make up >50% of the co-cultures. Imaging and transwell assay data (not included on poster) implicate cell-cell contact rather than paracrine factors as the primary driving factor of this phenotype. HER2 and PIK3CA inhibitors can effectively target the co-cultures and inhibiting the growth of the PIK3CA cells appears to halt the growth of the HER2 mutants as well. Conclusions: PIK3CA mutant cells can induce proliferation of quiescent HER2 mutant cells in growth-factor deprived conditions. This effect appears to the cell-cell contact mediated rather than driven by secreted factors. Co-cultures that contain PIK3CA and HER2 mutant cells have therapeutic vulnerabilities that can be exploited using drugs that are currently approved or in clinical trials. These results have important implications in understanding early tumor development and suggest that therapeutic approaches that target subclonal populations may be effective in certain tumors. Citation Format: Ian Waters, Swathi Karthikeyan, Lauren Dennison, David Chu, Ben Ho Park. Subclonal sociability: Interactions of HER2 and PIK3CAmutant cells in breast cancer [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 5149.

Abstract 46: 3D reconstruction and quantitative analysis of histology for prostate cancer

Abstract The integration of serial sectioning of tissue, digital whole slide imaging (WSI) and computational reconstruction algorithms enable the examination of histological samples in 3D at subcellular resolution. This allows visualizing and analyzing the imaged sample in its true three-dimensional context, offering a more comprehensive view on its spatial and morphological characteristics than that obtained via typical 2D examination. The advantages of reconstructing 3D models computationally using WSI over direct 3D imaging include the combination of high resolution, large sample sizes and compatibility with existing biochemical techniques such as in situ hybridization, immunohistochemistry and established histological staining and interpretation protocols. For this purpose, we developed a software pipeline to perform routine 3D reconstruction tasks for large image sizes and datasets in a fully automatic manner. The steps in our 3D reconstruction process are image acquisition, alignment of images to a shared coordinate space, and visualization of the reconstructed 3D data. We optimized algorithm selection and computationally intensive hyperparameter tuning using a quantitative benchmarking framework and Bayesian optimization. Further, we applied our existing pipeline for feature based analysis of tissue and extended it into 3D, allowing quantification and visualization of hundreds of features characterizing the tissue. We currently apply this system to characterize prostate cancer tumors. Prostate cancer is a heterogenous, often multifocal disease. In order to understand why and how certain tumor foci develop to a life-threatening disease over others, the tissue growth patterns and evolution of tumors with different genetic backgrounds need to be studied. We use mouse models of prostate cancer to study early tumor development connected to common genetic cancer alterations. By computing hundreds of features from the histology, and studying these in the spatial context, we gain important information of tumor characteristics as well as intra- and intertumor heterogeneity. In the future, we will further scale up the protocol to perform 3D reconstruction for serially sectioned human prostates in the near future. Citation Format: Pekka Ruusuvuori, Kimmo Kartasalo, Mira Valkonen, Masi Valkonen, Tapio Visakorpi, Matti Nykter, Leena Latonen. 3D reconstruction and quantitative analysis of histology for prostate cancer [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 46.

Effect of FGF/FGFR pathway blocking on lung adenocarcinoma and its cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are known to promote tumourigenesis through various mechanisms. Fibroblast growth factor (FGF)/FGF receptor (FGFR)-dependent lung cancers have been described. We have developed a mouse model of lung adenocarcinoma that was constructed through the induction of Fgf9 overexpression in type 2 alveolar cells. The expression of Fgf9 in adult lungs resulted in the rapid development of multiple adenocarcinoma-like tumour nodules. Here, we have characterised the contribution of CAFs and the Fgf/Fgfr signalling pathway in maintaining the lung tumours initiated by Fgf9 overexpression. We found that CAF-secreted Fgf2 contributes to tumour cell growth. CAFs overexpressed Tgfb, Mmp7, Fgf9, and Fgf2; synthesised more collagen, and secreted inflammatory cell-recruiting cytokines. CAFs also enhanced the conversion of tumour-associated macrophages (TAMs) to the tumour-supportive M2 phenotype but did not influence angiogenesis. In vivo inhibition of Fgfrs during early lung tumour development resulted in significantly smaller and fewer tumour nodules, whereas inhibition in established lung tumours caused a significant reduction in tumour size and number. Fgfr inhibition also influenced tumour stromal cells, as it significantly abolished TAM recruitment and reduced tumour vascularity. However, the withdrawal of the inhibitor caused a significant recurrence/regrowth of Fgf/Fgfr-independent lung tumours. These recurrent tumours did not possess a higher proliferative or propagative potential. Our results provide evidence that fibroblasts associated with the Fgf9-induced lung adenocarcinoma provide multiple means of support to the tumour. Although the Fgfr blocker significantly suppressed the tumour and its stromal cells, it was not sufficient to completely eliminate the tumour, probably due to the emergence of alternative (resistance/maintenance) mechanism(s). This model represents an excellent tool to further study the complex interactions between CAFs, their related chemokines, and the progression of lung adenocarcinoma; it also provides further evidence to support the need for a combinatorial strategy to treat lung cancer. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mistletoe and Immunomodulation: Insights and Implications for Anticancer Therapies.

In early tumor development, cancer cells develop a plethora of strategies to escape surveillance from the adaptive and innate immune system. Cancer immunotherapies, in particular immune checkpoint inhibitors, are becoming a highly promising cancer therapeutic approach that has remarkable increased progress in combating various cancer types. Unfortunately, their mechanisms of action induce some complications, such as inflammatory reactions and immune-related adverse events. In the management of side effects during anticancer therapy, complementary and integrative therapy approaches are becoming of growing interest. Particularly, mistletoe, Viscum album L. (VA), has a long traditional history of about 100 years as an add-on therapy of cancer treatment in German-speaking countries. Besides antitumoral and quality of life-promoting activities, VA applications reduce side effects of modern conventional anticancer therapies and exert immunomodulatory characteristics. As these properties may provide a good basis for a combination with modern oncological therapies, the biological activities of VA applications and mechanisms involved have to be understood. In this review, the impact of VA compounds on different cellular pathways and immunological reactions in the fight against cancerous cells is discussed.

Novel concept of the border niche: glioblastoma cells use oligodendrocytes progenitor cells (GAOs) and microglia to acquire stem cell-like features.

Glioblastoma (GBM) is a major malignant brain tumor developing in adult brain white matter, characterized by rapid growth and invasion. GBM cells spread into the contralateral hemisphere, even during early tumor development. However, after complete resection of tumor mass, GBM commonly recurs around the tumor removal cavity, suggesting that a microenvironment at the tumor border provides chemo-radioresistance to GBM cells. Thus, clarification of the tumor border microenvironment is critical for improving prognosis in GBM patients. MicroRNA (miRNA) expression in samples from the tumor, tumor border, and peripheral region far from tumor mass was compared, and five miRNAs showing characteristically higher expression in the tumor border were identified, with the top three related to oligodendrocyte differentiation. Pathologically, oligodendrocyte lineage cells increased in the border, but were rare in tumors. Macrophages/microglia also colocalized in the border area. Medium cultured with oligodendrocyte progenitor cells (OPCs) and macrophages induced stemness and chemo-radioresistance in GBM cells, suggesting that OPCs and macrophages/microglia constitute a special microenvironment for GBM cells at the tumor border. The supportive function of OPCs for GBM cells has not been discussed previously. OPCs are indispensable for GBM cells to establish special niches for chemo-radioresistance outside the tumor mass.

Modeling Melanoma in 3D uGel™‐based Artificial Tumor Tissues Demonstrate Initiation, Progression and Metastatic Processes In‐vitro

In a continuing collaboration between our laboratory, the UWRF Tissue and Cellular Innovation Center, and BRTI Lifesciences, LLC we have been evaluating the application of a commercial hydrogel as a matrix for the development and study of several artificial tumor tissues. Here we report on the application of uGel™, a recently developed chitosan/hyaluronate precast matrix to generate 3D melanoma artificial tissues. In this work, we coupled uGel™ matrix with mouse melanoma cell lines B16F10 and B16F1 to generate and study 3D tumor tissues in‐vitro. As we have previously reported with another earlier and related hydrogel, Cell‐Mate™, both of these cells lines successfully produced extensive and long‐lived tumor tissues. In addition, in this series of studies with uGel™ it was possible to model not just the end stages of tumor progression and metastasis, but also the early stages of tumor establishment and local invasion of the matrix in patterns which clearly reflect early tumor development in vivo. As we had seen before, the final stages of tumor development in melanoma includes the generation of two distinct populations of cells, one pigmented and one not. Additionally these two populations also correlate tightly with differential patterns of tissue formation ranging from highly branched and pigmented to completely rounded, smooth and lacking virtually any pigment. Although the branching features are clearly associated with local invasion, our work suggests that the pale, rounded features may actually represent the metastatic stage for these tumors. Interestingly, uGel™ based melanoma developed and progressed to full‐blown tumor masses within a three week period while in our earlier work with Cell‐Mate™ constructs fully developed tumors required from 2–3 months of long‐term culture to reach the same stage. Although the two materials are biochemically identical, they do differ in mechanical properties at the time of loading which may play a role in the significant differences observed in terms of developmental timeline. This study further support our view that small‐scale and long‐term 3D artificial tissues are an effective tool to better understand the biology of cancer and to study it's progression in‐vitro.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Using a variant of coverslip hypoxia to visualize tumor cell alterations at increasing distances from an oxygen source.

Early stages in tumor development involve growth in confined spaces, where oxygen diffusion is limited and metabolic waste products accumulate. This hostile microenvironment imposes strong selective pressures on tumor cells, leading eventually to the survival and expansion of aggressive subclones that condition further tumor evolution. To model features of this microenvironment in vitro, a diffusional barrier can be introduced in the form of a coverslip placed on top of cells, a method termed coverslip hypoxia. Using a variant of this method, with larger volume between coverslip and cells and with oxygen diffusion occurring only through a small hole in the center of the coverslip, we have visualized alterations in LNCaP tumor cells as a function of their distance to the oxygen source at the center. We observed remarkable morphological changes in LNCaP cells as the distance from the center increases, with cells becoming highly spread, displaying dynamic membrane protrusions and occasionally adopting a migratory phenotype. Concomitantly, cells farther from the center displayed marked increases in the hypoxia marker hypoxyprobe, whereas extracellular pH decreased in the same direction. Cells with altered morphology displayed prominent increases in fibrillar actin, as well as swollen mitochondria with distorted cristae and accumulation of neutral lipid-containing intracellular vesicles. These results show that an in vitro microenvironment that models diffusional barriers encountered by tumors in situ can have profound effects on tumor cells. The coverslip hypoxia variant we describe can be used to characterize in vitro the response of tumor cells to environmental conditions that play crucial roles in early tumor development.

Abstract P3-07-09: Tumour clonality in paired invasive breast carcinomas

Abstract Background: Multiple invasive breast tumours may represent either independent primary tumours or clonal recurrences of the first tumour, where the same progenitor cell gives rise to all of the detected tumours. Consequently, the driver events for the progenitor cell need to have been identical in early tumour development. Molecular classification of tumour clonality is not currently evaluated in multiple invasive breast carcinomas, despite evidence suggesting common clonal origins. Furthermore, there is no consensus about which type of biological data (e.g. copy number, mutation, histology) and especially which statistical method is most suitable to distinguish clonal recurrences from independent primary tumours. Methods: Thirty-seven invasive breast tumour pairs were stratified by laterality (bilateral vs. ipsilateral) and the time interval between the diagnoses of the first and second tumours (synchronous vs. metachronous). Both tumours from the same patient were analysed by integrating clinical characteristics (n = 37), DNA copy number (n = 37), DNA methylation (n = 8), gene expression microarray (n = 7), RNA sequencing (n = 3), and SNP genotyping data (n = 3). Different statistical methods, e.g. the diagnostic similarity index (SI), distance measure, shared segment analysis etc., were used to classify the tumours from the same patient as clonally related recurrences or independent primary tumours. Results: The SI applied on DNA copy numbers derived from aCGH (array comparative genomic hybridization) data was determined as the strongest indicator of clonal relatedness as it showed the highest concordance with all other methods. The distance measure was the most conservative method and the shared segment analysis most liberal. Concordant evidence for tumour clonality was found in 46% (17/37) of the patients. Notably, no significant association was found between the clinical characteristics and molecular tumour features. Conclusions: A more accurate classification of clonal relatedness between multiple breast tumours may help to mitigate treatment failure and relapse by integrating tumour-associated molecular features, clinical parameters, and statistical methods. In cases of extremely similar or different tumour pairs, the results showed consistency regardless of the method used. The SI can be easily integrated into clinical routine using FFPE samples to obtain copy number data. However, clinical guidelines with exact thresholds need to be defined to standardize clonality testing in a routine diagnostic setting. Citation Format: Biermann J, Parris TZ, Nemes S, Danielsson A, Engqvist H, Werner Rönnerman E, Forssell-Aronsson E, Kovács A, Karlsson P, Helou K. Tumour clonality in paired invasive breast carcinomas [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-07-09.

Genetic and Epigenetic Pathways of lethal (2) giant larvae Tumor Suppressor in Drosophila melanogaster

The tumor suppressors are fundamental development genes with a complete penetrance. Recessive mutations in the l(2)gl suppressor in drosophila are accompanied by complex phenotypic syndromes. Realization of the l(2)gl locus functions is performed by genetic and epigenetic pathways. The Lgl gene product in complex protein complexes controls the establishment of the cell polarity in epithelial stem and nerve cells. The degree of the l(2)gl involvement in the regulation of early development, longevity, stress response, and tumor development can depend on the microRNA expression level, the presence of intracellular symbionts, and mutations of other vital genes and is affected by various impacts.

Patterns of Tumor Progression Predict Small and Tissue-Specific Tumor-Originating Niches.

The development of cancer is a multistep process in which cells increase in malignancy through progressive alterations. Such altered cells compete with wild-type cells and have to establish within a tissue in order to induce tumor formation. The range of this competition and the tumor-originating cell type which acquires the first alteration is unknown for most human tissues, mainly because the involved processes are hardly observable, aggravating an understanding of early tumor development. On the tissue scale, one observes different progression types, namely with and without detectable benign precursor stages. Human epidemiological data on the ratios of the two progression types exhibit large differences between cancers. The idea of this study is to utilize data of the ratios of progression types in human cancers to estimate the homeostatic range of competition in human tissues. This homeostatic competition range can be interpreted as necessary numbers of altered cells to induce tumor formation on the tissue scale. For this purpose, we develop a cell-based stochastic model which is calibrated with newly-interpreted human epidemiological data. We find that the number of tumor cells which inevitably leads to later tumor formation is surprisingly small compared to the overall tumor and largely depends on the human tissue type. This result points toward the existence of a tissue-specific tumor-originating niche in which the fate of tumor development is decided early and long before a tumor becomes detectable. Moreover, our results suggest that the fixation of tumor cells in the tumor-originating niche triggers new processes which accelerate tumor growth after normal tissue homeostasis is voided. Our estimate for the human colon agrees well with the size of the stem cell niche in colonic crypts. For other tissues, our results might aid to identify the tumor-originating cell type. For instance, data on primary and secondary glioblastoma suggest that the tumors originate from a cell type competing in a range of 300 – 1,900 cells.

METCAM/MUC18 Decreases the Malignant Propensity of Human Ovarian Carcinoma Cells.

METCAM/MUC18 is an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family. It can carry out common functions of CAMs which is to perform intercellular interactions and interaction of cell with extracellular matrix in tumor microenvironment, to interact with various signaling pathways and to regulate general behaviors of cells. We and other two groups previously suggested that METCAM/MUC18 probably be utilized as a biomarker for predicting the malignant tendency of clinical ovarian carcinomas, since METAM/MUC18 expression appears to associate with the carcinoma at advanced stages. It has been further postulated to promote the malignant tendency of the carcinoma. However, our recent research results appear to support the conclusion that the above positive correlation is fortuitous; actually METCAM/MUC18 acts as a tumor and metastasis suppressor for the ovarian carcinoma cells. We also suggest possible mechanisms in the METCAM/MUC18-mediated early tumor development and metastasis of ovarian carcinoma. Moreover, we propose to employ recombinant METCAM/MUC18 proteins and other derived products as therapeutic agents to treat the ovarian cancer patients by decreasing the malignant potential of ovarian carcinoma.

Abstract PR09: Alterations in cell junctions and neuroendocrine differentiation are key early steps in Crebbp/Ep300 mutation-driven SCLC development

Abstract The abundance and heterogeneity of somatic alterations in small cell lung cancer (SCLC) present immense challenges in identifying key mechanisms of tumor development. Functional characterization of recurrent mutations is an essential step towards understanding the early stages of SCLC development, but this remains extremely difficult due to the scarcity of relevant and tractable models for defining oncogenic drivers and molecular pathways. In a previous study, we derived precancerous cells (preSCs) from the Rb/p53/Rbl2-mutant mouse model of SCLC and developed an engineered preSC-based model of early tumor development. In this model, activation of oncogenic drivers transforms preSCs into tumorigenic cells that form spheroid clusters characteristic of SCLC in culture and subcutaneous tumors in athymic nude mice. Using this streamlined approach, we found that both retroviral amplification of the Myc family members and CRISPR/Cas9-mediated mutation of Crebbp/Ep300 were sufficient to cause transformation of preSCs into SCLC cells. Notably, both sets of mutant preSC underwent a similar phenotypic change from adherent monolayers to spheroid aggregates, suggesting that the two different types of alterations interact with or converge on common molecular pathways. Comparative profiling of control preSCs and mutant preSCs carrying either a Myc family amplification or Crebbp/Ep300 mutation revealed that alterations in cell junctions and neuroendocrine differentiation are the most significant molecular changes during cellular transformation. The transformed preSCs exhibited decreased expression of adhesion-related genes, including Cdh1 and Dsp, but increased expression of mesenchymal genes Vim and Twist1. The transformed cells also showed reduced expression of neuroendocrine (NE) differentiation genes, including Ascl1, Chga, and Syp. Furthermore, these changes coincided with increased expression of Notch receptors and decreased expression of their ligands Dlls and Jags. Taken together, these novel findings suggest that a phenotypic switch from Dll/Jag-expressing NE cells to Notch-expressing non-NE cells is a key event in early SCLC development. This study also demonstrated that the preSC-based model can facilitate functional validation of recurrent mutations in SCLC and molecular pathways that will be further explored for mechanistic elucidation and development of targeted therapies. This abstract is also being presented as Poster A09. Citation Format: Dong-Wook Kim, Keebeom Kim, Colin T. Dunn, Kwon-Sik Park. Alterations in cell junctions and neuroendocrine differentiation are key early steps in Crebbp/Ep300 mutation-driven SCLC development [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR09.

PO-220 Defining the role of heparanase in breast cancer progression using the PyMT-MMTV mouse model

IntroductionHeparanase (HPSE) is a beta-d-endoglucuronidase and the only mammalian enzyme that cleaves heparan sulphate (HS), a major structural and regulatory component of the extracellular matrix (ECM) and the vascular basement membrane (BM). The expression of HPSE is tightly controlled and under physiological conditions is limited to immune cells, endothelial cells, placental trophoblasts and keratinocytes. However, during pathological conditions such as cancer, HPSE expression is dysregulated and high expression often correlates with poor patient survival. The cleavage of HS by HPSE expressing tumours degrades the ECM/BM and releases HS bound growth factors and cytokines, promoting cellular signalling in a positive feed-back mechanism. This in turn leads to enhanced primary tumour growth, metastasis, angiogenesis and inflammation. HPSE is therefore a critical promoter of the hallmarks of cancer and has generated significant interest as an anti-cancer drug target. However, despite decades of research, the precise mechanistic role of HPSE in the tumour microenvironment remains poorly defined. Breast cancer is the most prevalent malignancy in women worldwide. Clinical data reveals that the expression of HPSE in mammary tumours results in poor patient survival.Material and methodsWe recently generated a HPSE-deficient C57Bl/6 mouse strain (C57Bl/6xHPSE-/-) that were crossed with spontaneous mammary tumour developing PyMT-MMTV mice to generate the PyMT-MMTVxHPSE-/- strain, providing us with a valuable in vivo model to characterise the role of HPSE in early mammary tumour development, tumour progression and metastasis.Results and discussionsOur data indicate that although HPSE promoted tumour angiogenesis, overall tumour development and metastasis between PyMT-MMTV and PyMT-MMTVxHPSE-/- mice remained comparable. By examination of the tumour microenvironment, we also demonstrate that the tumour stroma positively contributes to mammary tumour HPSE activity, promoting tumour angiogenesis. In contrast, HPSE expressed in the tumour-bearing host appeared to play no significant role in tumour growth and metastasis. These data suggest that in the PyMT-MMTV model and indeed, in certain cancer settings such as breast cancer, HPSE may not play as significant a role to what has been proposed over the last two decades.ConclusionThese findings may have important implications for the ongoing development and application of HPSE inhibitors in the treatment of cancer.

Serum MMP-8 and TIMP-1 predict prognosis in colorectal cancer

BackgroundAlmost all of the extracellular matrix (ECM) components can be degraded by the endoproteinases matrix metalloproteinases (MMPs). Important regulators of MMPs, and thereby of the extracellular environment, are tissue inhibitors of metalloproteinases (TIMPs), and especially TIMP-1. Early tumor development, as well as distant metastasis, may be results of an MMP/TIMP ratio imbalance altering the ECM. MMPs are elevated in several inflammatory conditions. Our aim is to investigate the prognostic role of MMP-8, − 9, and TIMP-1 in colorectal cancer (CRC) and their relationship to inflammation.MethodsWe included 337 colorectal cancer patients and 47 controls undergoing surgery at Helsinki University Hospital in Finland, 1998–2011. Serum levels of MMP-8 and plasma levels of C-reactive protein (CRP) were determined with a time-resolved immunofluorometric assay (IFMA), and MMP-9 and TIMP-1 with commercial enzyme-linked immunosorbent assay (ELISA) kits. Association and correlation analyses were performed with the Mann-Whitney U, Kruskal-Wallis, and Spearman rank correlation tests. Survival curves were constructed according to the Kaplan-Meier method and compared with the log-rank test.ResultsAmong patients with advanced disease, serum levels of MMP-8 and TIMP-1 were elevated. CRC patients with high MMP-8 (HR (hazard ratio) 1.72, 95% confidence interval (CI) 1.17–2.52, P = 0.005) and those with high TIMP-1 (HR 1.80, 95% CI 1.23–2.64, P = 0.002) had worse prognoses. MMP-9 level failed to serve as a prognostic factor. In multivariable survival analysis, Dukes stage, and low MMP-9/TIMP-1 molar ratio (HR 0.46, 95% CI 0.33–0.98, P = 0.042) were independently predicted prognosis. A weak correlation between CRP and MMP-8 (rS = 0.229, P < 0.001), and TIMP-1 (rS = 0.280, P < 0.001) was noted. Among patients showing no systemic inflammatory response, MMP-8 (HR 1.66, 95% CI 1.10–2.53, P = 0.017) and TIMP-1 (HR 1.59, 95% CI 1.05–2.42, P = 0.029) were prognostic factors.ConclusionsMMP-8 and TIMP-1 in serum, but not MMP-9, identified CRC patients with bad prognosis. Among patients showing no systemic inflammatory response, MMP-8 and TIMP-1 may associate with poor prognosis.

IL17A Regulates Tumor Latency and Metastasis in Lung Adeno and Squamous SQ.2b and AD.1 Cancer.

Somatic mutations can promote malignant transformation of airway epithelial cells and induce inflammatory responses directed against resultant tumors. Tumor-infiltrating T lymphocytes (TIL) in early-stage non-small cell lung cancer (NSCLC) secrete distinct proinflammatory cytokines, but the contribution of these TILs to tumor development and metastasis remains unknown. We show here that TILs in early-stage NSCLC are biased toward IL17A expression (Th17) when compared with adjacent tumor-free tissue, whereas Th17 cells are decreased in tumor infiltrating locoregional lymph nodes in advanced NSCLC. Mice in which Pten and Smad4 (Pts4d/d ) are deleted from airway epithelial cells develop spontaneous tumors, that share genetic signatures with squamous- (SQ.2b), and adeno- (AD.1) subtypes of human NSCLC. Pts4d/d mice globally lacking in IL17a (Pts4d/dIl17a-/- ) showed decreased tumor latency and increased metastasis. Th17 cells were required for recruitment of CD103+ dendritic cells, and adoptive transfer of IL17a-sufficient CD4+ T cells reversed early tumor development and metastasis in Pts4d/dIl17a-/- mice. Together, these findings support a key role for Th17 cells in TILs associated with the Pts4d/d model of NSCLC and suggest therapeutic and biomarker strategies for human SQ2b and AD1 lung cancer. Cancer Immunol Res; 6(6); 645-57. ©2018 AACR.

The Evolutionary Landscape of Localized Prostate Cancers Drives Clinical Aggression

The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospectivestudies of low-risk tumors suitable for active surveillance.

Model Prediction and Validation of an Order Mechanism Controlling the Spatiotemporal Phenotype of Early Hepatocellular Carcinoma

Recently, hepatocyte–sinusoid alignment (HSA) has been identified as a mechanism that supports the coordination of hepatocytes during liver regeneration to reestablish a functional micro-architecture (Hoehme et al. in Proc Natl Acad Sci 107(23):10371–10376, 2010). HSA means that hepatocytes preferentially align along the closest micro-vessels. Here, we studied whether this mechanism is still active in early hepatocellular tumors. The same agent-based spatiotemporal model that previously correctly predicted HSA in liver regeneration was further developed to simulate scenarios in early tumor development, when individual initiated hepatocytes gain increased proliferation capacity. The model simulations were performed under conditions of realistic liver micro-architectures obtained from 3D reconstructions of confocal laser scanning micrographs. Interestingly, the established model predicted that initiated hepatocytes at first arrange in elongated patterns. Only when the tumor progresses to cell numbers of approximately 4000, does it adopt spherical structures. This prediction may have relevant consequences, since elongated tumors may reach critical structures faster, such as larger vessels, compared to a spherical tumor of similar cell number. Interestingly, this model prediction was confirmed by analysis of the spatial organization of initiated hepatocytes in a rat liver tumor initiation study using single doses of 250 mg/kg of the genotoxic carcinogen N-nitrosomorpholine (NNM). Indeed, small clusters of GST-P positive cells induced by NNM were elongated, almost columnar, while larger GDT-P positive foci of approximately the size of liver lobuli adopted spherical shapes. From simulations testing numerous possible mechanisms, only HSA could explain the experimentally observed initial deviation from spherical shape. The present study demonstrates that the architecture of small cell clusters of hepatocytes early after initiation is still controlled by physiological mechanisms. However, this coordinating influence is lost when the tumor grows to approximately 4000 cells, leading to further growth in spherical shape. Our findings stress the potential importance of organ micro-architecture in understanding tumor phenotypes.

The Role of Natural Killer T Cells in Cancer-A Phenotypical and Functional Approach.

Natural killer T (NKT) cells are a subset of CD1d-restricted T cells at the interface between the innate and adaptive immune system. NKT cells can be subdivided into functional subsets that respond rapidly to a wide variety of glycolipids and stress-related proteins using T- or natural killer (NK) cell-like effector mechanisms. Because of their major modulating effects on immune responses via secretion of cytokines, NKT cells are also considered important players in tumor immunosurveillance. During early tumor development, T helper (TH)1-like NKT cell subsets have the potential to rapidly stimulate tumor-specific T cells and effector NK cells that can eliminate tumor cells. In case of tumor progression, NKT cells may become overstimulated and anergic leading to deletion of a part of the NKT cell population in patients via activation-induced cell death. In addition, the remaining NKT cells become hyporesponsive, or switch to immunosuppressive TH2-/T regulatory-like NKT cell subsets, thereby facilitating tumor progression and immune escape. In this review, we discuss this important role of NKT cells in tumor development and we conclude that there should be three important focuses of future research in cancer patients in relation with NKT cells: (1) expansion of the NKT cell population, (2) prevention and breaking of NKT cell anergy, and (3) skewing of NKT cells toward TH1-like subsets with antitumor activity.

Somatic evolutionary timings of driver mutations

BackgroundA unified analysis of DNA sequences from hundreds of tumors concluded that the driver mutations primarily occur in the earliest stages of cancer formation, with relatively few driver mutation events detected in the late-arising subclones. However, emerging evidence from the sequencing of multiple tumors and tumor regions per individual suggests that late-arising subclones with additional driver mutations are underestimated in single-sample analyses.MethodsTo test whether driver mutations generally map to early tumor development, we examined multi-regional tumor sequencing data from 101 individuals reported in 11 published studies. Following previous studies, we annotated mutations as early-arising when all tumors/regions had those mutations (ubiquitous). We then inferred the fraction of mutations occurring early and compared it with late-arising mutations that were found in only single tumors/regions.ResultsWhile a large fraction of driver mutations in tumors occurred relatively early in cancers, later driver mutations occurred at least as frequently as the early drivers in a substantial number of patients. This result was robust to many different approaches to annotate driver mutations. The relative frequency of early and late driver mutations varied among patients of the same cancer type and in different cancer types. We found that previous reports of the preponderance of early driver mutations were primarily informed by analysis of single tumor variant allele profiles, with which it is challenging to clearly distinguish between early and late drivers.ConclusionsThe origin and preponderance of new driver mutations are not limited to early stages of tumor evolution, with different tumors and regions showing distinct driver mutations and, consequently, distinct characteristics. Therefore, tumors with extensive intratumor heterogeneity appear to have many newly acquired drivers.