Received: 11 February 2020; Accepted: 12 March 2020; Published: 14 March 2020 Abstract: Experimental models of neuroendocrine tumor disease are scarce, with only a few existing neuroendocrine tumorcell lines of pancreatic origin (panNET). Their molecular characterization has so far focused on the neuroendocrine phenotype and cancer-related mutations, while a transcription-based assessment of their developmental origin and malignant potential is lacking. In this study, we performed immunoblotting and qPCR analysis of neuroendocrine, epithelial, developmental endocrine-related genes as well as next-generation sequencing (NGS) analysis of microRNAs (miRs) on three panNET cell lines, BON-1, QGP-1, and NT-3. All three lines displayed a neuroendocrine and epithelial phenotype; however, while insulinoma-derived NT-3 cells preferentially expressed markers of mature functional pancreatic β-cells (i.e., INS, MAFA), both BON-1 and QGP-1 displayed high expression of genes associated with immature or non-functional β /δ-cells genes (i.e., NEUROG3), or pancreatic endocrine progenitors (i.e., FOXA2). NGS-based identification of miRs in BON-1 and QGP-1 cells revealed the presence of all six members of the miR-17–92 cluster, which have been implicated in β-cell function and differentiation, but also have roles in cancer being both oncogenic or tumor suppressive. Notably, both BON-1 and QGP-1 cells expressed several miRs known to be negatively associated with epithelial–mesenchymal transition, invasion or metastasis. Moreover, both cell lines failed to exhibit migratory activity in vitro.
and detection of ZEB1, SNAIL and Vimentin can be difficult and confounded by labelling of stromal cells (Francí et al., 2009; Spaderna et al., 2006; Toiyama et al., 2013), we propose that PBX3 may be a useful marker to highlight and further study colon cancer cells undergoing EMT in situ. Furthermore, we demonstrate that PBX3 mRNA levels strongly correlated with EMT in a large gene expression data set derived from 1,032 colon cancer samples. Considering the restriction of PBX3 expression to cancer cells, we therefore propose that on the gene expression level PBX3 may indicate the overall degree of EMT in colon cancer specimens with little confounding by the amount of stromal tissue within each sample. Of note however, PBX3 expression was not completely restricted to infiltrative tumor cells at the leading tumor edge but also extended to glandular differentiated colon cancer cells, especially in cases with high levels of PBX3 expression. Because similar observations also were made for ZEB1 and SNAIL (Francí et al., 2009; Spaderna et al., 2006), it remains to be determined to what extent infiltrative tumorcell morphology and EMT related factors indicate identical or only partially overlapping colon cancer cell subpopulations.
In the beginning of the 1900s Paul Ehrlich introduced the concept of drugs to treat infectious diseases and the newly coined word “chemotherapy”, delineating it as the use of chemical compounds for disease treatment. Furthermore, he projected the idea of the “magic bullet” aimed at killing a harmful agent while leaving healthy tissues untouched. [4, 5] In the 1960s, combination of surgery and radiotherapy was acknowledged as standard cancer treatment.  However, only one third of treated patients were treated successfully as the applied therapies were not able to handle small metastases.  At that time, new research remarkably showed that a combination of classical methods with chemotherapy can lead to full cancer remission in patients with various tumors.  The first applied chemotherapeutic agents tested in humans comprised toxic nitrogen mustards, chlorambucil, and cyclophosphamide targeting DNA by alkylation (Figure 1).  Further, antifolates like methotrexate were introduced as higher proliferation rates for tumors treated with folic acid were observed.  Early in the following years chemotherapy became the predominant approach in tumor therapy. [6-8] Hence, a plethora of cancer targeting compounds was designed. Today, the majority of chemotherapeutics follow a non- specific uptake through lipophilic interactions with the cell membrane of the tumorcell.  Usually, these compounds promote killing of rapidly dividing cells exhibiting higher proliferations rates. [6, 10] This is mediated by inhibiting microtubule function, DNA synthesis or protein function for example.  However, not only malignant cells are affected but numerous healthy ones, for example those from the epithelium, bone marrow and gastrointestinal tract. [6, 12] Additionally, DNA synthesis interfering nucleoside analogues (thioguanine, cytosine arabinoside), DNA interacting agents such as anthracyclines and actinomycin D, and tubulin targeting
Although tumorcell-TAM crosstalk is dependent on many factors, secreted factors (such as cytokines, chemokines, etc.) play a significant role in the crosstalk. Cytokines and chemokines are low molecular weight proteins, mainly produced by macrophages and lymphocytes. They mediate intra- and extra- cellular communication as hormones and neurotransmitters through an autocrine, paracrine, and endocrine manner. Upon binding to specific cell surface receptors, they regulate a variety of cellular processes, such as local and systemic anti- and pro- inflammation, cellular proliferation, metabolism, chemotaxis, and tissue repair, etc. In the TME, the primary role of these factors is to regulate the tumor immunity cycle. Cytokines and chemokines produced by tumor-infiltrating immune cells play a significant role in tumor development, progression, metastasis, and therapy resistance; therefore, they widely used as diagnostic and prognostic biomarkers in the treatment of cancer. As shown in Table 1, most common cytokines and chemokines used in the therapeutic management of lung cancer are IL6, tumor necrosis factor α (TNFα), IL10, IFNγ, IL2, IL22, IL32, IL37, IL8, CCL2, C-X3-C motif chemokine ligand (CX3CL1) ( 53 – 59 , 64 , 82 – 85 ), among which macrophages are the major source of IL6, TNFα, IL10, IL8, CCL2, and CX3CL1 [( 86 , 87 ); Figure 1]. CCL2 and CX3CL1 receive special attention in chemokine biology, because of their unique phenotypic and functional properties. The decades of extensive research in the field of cytokines and chemokines in cancer development published outstanding research and review articles. Therefore, in this review, we summarized the published literature from the year 2000 to the year 2019, specifically focusing on the role of IL6, TNFα, IL10, CCL2, CX3CL1, IL8 in the macrophages-tumor cells crosstalk; leading to lung cancer development and progression.
DNA damage response is triggered when sensor proteins ATM (ataxia telangiectasia mutated) and ATR (also called ataxia telangiectasia and Rad3-related protein) detect structural distortions or breaks . After DNA damage, CHK2 is phosphorylated by ATM on the priming site T68, and in turn, phosphorylates more than 24 proteins to induce apoptosis, DNA repair, or tolerance of the damage . In wildtype cells, CHK2 phosphorylates Rb which enhances the formation of the transcriptionally-inactive pRb/E2F-1 complex causing G1/S arrest and suppression of apoptosis. Pronounced activation of CHK-2 in NCI-H526 and A549 cells indicates direct damage of DNA by fascaplysin and activation of the corresponding cellular responses in both cell lines. The cyclic AMP response element-binding protein (CREB) initiates transcriptional responses associated with cell survival to a wide variety of stimuli following its phosphorylation on Ser-133. Whereas fascaplysin treatment resulted in decreased phosphorylation of CREB in NCI-H526 cells, this transcription factor is hyperphosphorylated in A549 cells, possibly indicating anti- and pro-survival signaling, respectively [33,34]. Furthermore, cisplatin-induced activation of FAK has been linked to increased chemoresistance in ovarian cancer cells and FAK inhibitors induce tumorcell apoptosis . Activated FAK forms a complex with Src family kinases and seems to provide a prosurvival signal in NCI-H526 cells, in contrast to fascaplysin-treated A549 cells . In addition, overexpression of Src in cancer accelerates metastasis and is responsible for chemoresistance via multiple downstream signaling pathways, concerning Akt, MAPKs, STAT3, cytokines, etc. . Therefore, activation of a
Metastasis-initiating capabilities of CTCs based on stemness properties are difficult to detect since CTCs show extensive het- erogeneity and only an extremely small fraction of these cells is able to establish secondary lesions [ 32 , 33 ]. In vitro expansion of relevant CTCs has been reported for only a limited number of tumors and cell lines so far [ 34 ]. We have obtained five perma- nent CTC cell lines from blood samples of patients bearing ex- tended disease small cell lung cancer (ED-SCLC) [ 35 ]. Small cell lung cancer (SCLC) comprises approximately 15% of all lung cancers and is found disseminated in the great majority of patients at first presentation [ 36 ]. Patients respond well to first- line platinum-based combination therapy with response rates on the order of 70%–90% in limited disease and 50%– 60% in extended disease [ 36 , 37 ]. However, nearly all patients with SCLC eventually relapse with chemo- and radioresistant tumors which are difficult to treat and have a dismal prognosis [ 38 ]. These characteristics of SCLC suggest that it may be enriched in CSCs and the general resistance is effected by a CSC subpop- ulation [ 39 , 40 ]. In the present study, the SCLC CTC and tumorcell lines were used to study chemosensitivity to the CSC- targeting drugs salinomycin and niclosamide and to characterize their expression of selected CSC markers and pluripotent tran- scription factors.
Tumorcell spread to distant sites is a complex process involving multiple cell types, soluble growth factors, adhesion receptors, and tissue remodeling [ 1 , 27 ]. Pericellular proteases are involved in cancer invasion and metastasis due to their ability to degrade ECM constituents [ 11 , 12 ]. Furthermore, proteases regulate progression and dissemination through processing of cell adhesion molecules, cytokines, growth factors, and kinases [ 1 ]. SCLC is surrounded by an extensive stroma of ECM, protecting cancer cells by prosurvival signaling [ 28 – 30 ]. Although several enzymes of the proteolytic tumor network are associated with invasion and metastasis, the proteases responsible for the migration and invasion of CTCs have not been identified so far. CTCs are highly heterogeneous and only a small fraction of these cells is capable of inducing metastases [ 9 , 15 ]. Availability of two CTC cell lines established from SCLC enabled us to screen the proteases secreted by these tumor cells in vitro. Both CTC lines used were in tissue culture for several months after initiation of the lines. However, according to their transcriptomic and proteomic profile as well as biomarkers and morphology (formation of spheroids), these lines exhibit a stable phenotype. This Western blot screen comprised 35 proteases including ADAMs 8, 9, S1, and S13; cathepsins A, B, C, D, E, L, S, V, and X/Z/P; MMPs 1, 2, 3, 7, 8, 9, 10, 12, 13; kallikreins 5, 6, 7, 10, 11, 13; neprilysin/CD10, presenilin-1, PC-9, proteinase 3, and uPA. Of all these enzymes, uPA, MMP-8 and -9 as well as several cathepsins were expressed in SCLC tumor lines, the two SCLC CTCs and conditioned macrophages in our screening experiments. Longitudinal biopsies are rarely available for SCLC patients. However, a series of three cell lines, namely GLC14, GLC16, and GLC19, were established from the biopsies of a single SCLC patient [ 24 ]. In detail, the GLC14 cell line was from a right supraclavicular node metastasis of
68 and NOTCH activity through FRA1 and NICD was most discriminatory in predicting patient outcome and tumor metastasis. Importantly however, since patients whose tumors showed low activity for both pathways survived best and showed lowest tumor progression and metastasis rates, this further strengthened the rationale for combined targeted treatment against both pathways. As immunostainings for FRA1 and NICD readily indicated presence and extent of respective tumorcell subpopulations in colon cancer specimens, and also often were consistent in primary colon cancers and their metastases, these may well be evaluated as predictive biomarkers. The stratification of CRC patients according to FRA1 and NICD expression in future clinical trials, might help to identify patients that benefit from combinatorial therapies with MEK and γ-secretase inhibitors 27,49 . Consequently, the
A careful review of the existing evidence suggests that tumor cells can acquire and develop resistance to apoptosis in several ways [35, 50, 51]. One of the commonly discussed strategies leading to apoptotic resistance is the loss of p53 tumor suppressor protein function due to mutation. Liu, Zhang, and Feng noted that the p53 tumor suppressor protein is known to promote apoptosis by activating proapoptotic Bcl-2 proteins. Additionally, it was observed that p53 is a fundamental tumor suppressor due to its antioxidant defense mechanism and regulation of energy metabolism. Tumor-associated mutant p53 proteins not only lose their tumor suppressive function but also acquire oncogenic capabilities, such as the promotion of tumorcell survival and proliferation and the control of metabolic changes, metastasis, and angiogenesis [52, 53, 54]. Dysregulated death receptor pathways have also been linked to apoptosis resistance. For example, absence of the Fas receptor protects tumor cells from immune destruction mediated by cytotoxic lymphocytes. In other cases, overexpression of c-FLIP in cancer patients protects tumor cells from cytotoxic T cell-induced apoptosis [55, 56, 57].
The human PIWI subfamily comprises HIWI (PIWI-LIKE 1), HILI (PIWI-LIKE 2), HIWI3 (PIWI-LIKE 3) and HIWI2 (PIWI-LIKE 4). PIWI-LIKE 2 is exclusively expressed in spermatogonia and pre-meiotic spermatocytes ( Sasaki et al., 2003 ). However, it has been demonstrated to be temporarily activated in somatic cells in response to DNA damages ( Lim et al., 2013 ) Furthermore, PIWI-LIKE 2 reveals ectopic expression in several tumor entities, and its intragenically activated products, such as PL2L60A, are expressed in various types of tumorcell lines ( Ye et al., 2010 ; Gainetdinov et al., 2014 ). Potential regulation mechanisms of PIWI-LIKE 2 expression are scarcely investigated. Normally, MILI (the murine homolog of HILI/PIWI-LIKE 2) is exclusively expressed in the spermatogonia and spermatocytes ( Sasaki et al., 2003 ) and in the female oocytes and supporting cells ( Lim et al., 2013 ).
Prior to immunostaining, slides were deparrafined using a decreasing alcohol dilution series. Later, heat-mediated antigen retrieval was performed and unspecific binding sides were reduced using Protein block (Dako by Agilent Technologies, Santa Clara, CA, USA). Vasculature was stained using a 1:100 dilution of anti-CD31 (ab28364)) and 1:100 fluorescent Alexa 546 anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Hypoxia was detected via bound pimonidazole adducts using a 1:100 mouse –anti-pimonidazole–FITC (Hypoxyprobe, HP6-100Kit). Additionally, tissue sections were stained with Hoechst 33342 (Life Technologies by Thermo Fisher, Waltham, MA, USA) to label cell nuclei. Slides were imaged for fluorescence on the ImageXpress Micro widefield imaging system (Molecular Devices, Sunnyvale, CA, USA) with 10 × air objective and attached CCD camera.
RNA-Isolation, microarray analysis and real-time PCR. Total RNA was isolated from three independent human neuroblastoma tissue samples (see above) or 2D, 3D-static and 3D bioreactor neuroblastoma cell cultures (each in triplicate), using the RNeasy MiniKit (Qiagen). For microarray analysis, 1 μg RNA per sample was used. Gene expression analysis was performed at the house- internal Genomics and Proteomics Core Facility using human whole genome HT-12 v4 BeadChips. Normalization of the raw intensity data was performed by the microarray unit of the DKFZ Genomics and Proteomics Core Facility with Illumina BeadStudio Data Analysis Software version v4_r2. The normalized gene expression profiles were further analyzed by principal components analysis (PCA) using the statistical software R. 68 For analysis of autophagy transcription factor expression the following probesets were applied: FOXO3: ILMN_1844692; HIF1A: ILMN_2379788; MITF: ILMN_2304186; MYCN: ILMN_1653761; NFE2L2: ILMN_1790909; TFE3: ILMN_1764826; TFEB: ILMN_1733616; SDHA: ILMN_1744210. Real-time RT-PCR was performed as described previously with at least three biological replicates and two technical replicates. 66 Data were normalized against neuroblastoma house- keeping genes SDHA and HPRT 69 and set in relation to negative control. The following specific primer pairs were used: ABL1 (forward: 5 ′-TTGACCAAGCCTCT ACAGGG-3 ′, reverse: 5′-AGACCCGGAGCTTTTCACCT-3′), ATG3 (forward: 5′-GA CCCCGGTCCTCAAGGA A-3 ′, reverse: 5′-TGTAGCCCATTGCCATGTTGG-3′), ATG16L2 (forward: 5 ′-TGGACAAGTTCTCAAAGAAGCTG-3′, reverse: 5′-CCTAGT GCGACCAGTGAT-3 ′), HDAC6 (forward: 5′-CAAGGAACACAGTTCACCTTCG-3′, reverse: 5 ′-GTTCCAAGGCACATTGATGGTA-3′), HDAC10 (Primer #1: forward: 5 ′-CTCACTGGAGCTGTGCAAAA-3′, reverse: 5′-GATCCTGTGTAGCCCGTGTT-3′; Primer #2: forward: 5 ′-ATCTCTTTGAGGATGACCCCAG-3′, reverse: 5′-ACTGCGT CTGCATCTGACTCTC-3 ′; Primer #3: forward: 5′-CAGTTCGACGCCATCTACTT C-3 ′, reverse: 5′-CAAGCCCATTTTGCACAGCTC-3′), HPRT (hypoxanthine phos
Immunohistochemistry of the tumorosphere sections. Cluster sections were deparaffinized at 60 °C for 25 min- utes. Afterwards, rehydration took place by incubation for 3 × 10 minutes with xylol and five minute incubations with absolute, 96%, 70%, 50%, 30% ethanol and PBS. Antigens were retrieved by a 20-min incubation in hot EDTA buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH = 9) in a steamer. After 20 minutes cooling at room temperature and 2 × 5 minutes washing with PBS, clusters were permeabilized with PBS-Tween 0,2% for five minutes. Thereafter, unspecific background was blocked with 5% FCS for 30 minutes at room temperature and primary antibody incubation for CHGA (Acris Antibodies AP15478PU-T, 1:300), CAIX (Novus Biologicals NB100-417, 1:200), CD56 (Biolegend MEM-188, 1:100) and Ki-67 (Ventana 790-4286, 1:3) diluted in 0,1% BSA took place in a humidity chamber at room temperature for one hour. Slides were washed 3 × 10 minutes in PBS-Tween 0,05% and corresponding Envision + HRP labelled polymer antibodies (Dako, Glostrup, Denmark) were applied to the slides for 30 minutes at room temperature in a humidity chamber. After 3 × 10 minutes wash- ing steps with PBS-Tween 0,05%, visualization took place by adding DAB chromogen (Dako). Reaction was stopped by immersing into distilled water and cell nuclei were counterstained with hematoxylin (Dako). After eight seconds in PBS, and rinsing with distilled water, sections were mounted with Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Aquisition of the microscopic images and quantitiative evaluation was done using the TissueFAXS System and HistoQuest software 184.108.40.206 (TissueGnostics, Vienna, Austria).
To overcome these limitations of EpCAM, we decided to test CD147, CA9, and CD70 as potential tumor markers for ccRCC. CD147 has already been well described as a tumor-specific protein in various tumor entities [ 58 – 65 ]. This transmembrane glycoprotein is highly homologous to proteins of the immunoglobulin (Ig) superfamily. CD147 is involved in matrix degeneration, tumorcell invasion, metastasis, and angiogenesis via the regulation of glycosylation and the induction of proteinases [ 58 ]. Both CD147 and its ligand MMP-9 (matrix metalloprotease-9) are overexpressed in RCC [ 59 , 60 ]. CD147 upregulation in tumor cells is associated with poor prognoses [ 61 , 62 ]. In addition, CD147 is overexpressed in patients treated with sunitinib therapy and in sunitinib-resistant 786-O cells [ 63 ]. In line with other studies [ 64 , 65 ], our data suggest that parts of normal tissues express CD147 as well. However, the expression was strong in the tumor-associated exosomes, supporting its role as a putative marker for RCC-released exosomes. The expression of CD147 in normal cell exosomes has to be further evaluated.
The HIV/SIV accessory protein Nef is known to down-modulate cell surface receptors that are required for virus entry such as CD4, CCR5 and CXCR4 to block lethal viral superinfection of the infected cell. The chemokine receptor CXCR4 also plays an important role in promoting tumorcell proliferation, metastasis and angiogenesis. Therefore it was of interest to evaluate if Nef can down-regulate CXCR4 in tumor cells since this could affect these critical prognostic parameters. The CXCR4 + tumorcell line HeLa-ACC was transfected with Nef from SIVmac239 and cell surface expression of the receptor was monitored by FACS analysis. Real-time cellular analysis was performed and cell migration was evaluated by an in vitro scratch assay. The in vitro tube formation assay was carried out on Nef transfected HUVEC cells to evaluate if Nef affects angiogenesis. Also, the influence of Nef on intracellular signaling was evaluated by Western blot analysis. Additionally, COS-7 cells were co-transfected with Nef and CXCR4 or its transcript variant, CXCR4-Lo, and were treated in the same way as described for HeLa-ACC.
Our results further suggest that esophageal carcinoma, like melanoma, could exploit the PD1 pathway to promote cancer progression both by dampening tumor-specific immunity via engagement of TIL-expressed PD1 and by triggering tumorcell-intrinsic growth signals via engage- ment of cancer cell-expressed PD1. However, whether PD1 does indeed modulate the antitumor immune response and/ or function as a tumorcell-intrinsic growth receptor in this malignancy requires future dedicated studies. In a separate study, 13 PD1 and PD-L1 expression were assessed previ- ously in tumor biospecimens from 349 patients with esophageal squamous cell carcinoma, and PD-L1 expres- sion levels were found to correlate significantly with favorable outcome, whereas PD1 expression within the TME did not show any significant associations with clini- copathologic parameters. 13
In vitro proliferation of transformed cell lines only partially reflects in vivo tumor formation. Within an organism tumor cells are exposed to a variety of cytokines that may affect survival and proliferation. Importantly the majority of these cytokines signals via the JAK/STAT cascade. STAT3 mediates target gene expression downstream of pro-inflammatory cytokines such as IL-6, TNF-α or IL-10 . We thus speculated that despite the unaltered tumorcell proliferation in vitro, there may be a difference in tumor growth in vivo. To test this, Stat3 fl/fl and Stat3 ∆/∆ BCR/ABL p185+ cells were injected subcutaneously into the flanks of C57BL/6 wild-type mice. Intriguingly, after 11 days STAT3-deficient lymphoma sizes were significantly increased compared to wild-type tumors. The increased tumor size was evident irrespective whether the STAT3 abrogation was achieved genetically (Figure 3A) or by lentiviral knockdown (Figure 3B).
The first description of CTCs in the peripheral blood of patients with solid malignancies dates from the 19 th century (4). A fraction of these CTCs presumably derives from the primary tumor as one of the initial steps in the process of metastasis, whereas the remaining CTCs probably originate from metastatic cancers. Since the likely outcome of cancer patients is largely determined by the metastatic potential of the primary tumor and the number of CTCs is likely to reflect its aggressiveness, CTC detection and quantification are considered to bear great potential as a prognostic marker in oncology (5). Indeed, CTCs detected in peripheral blood of patients with solid epithelial tumors, e.g. breast (6,7), colorectal (8,9) and prostate (10,11) cancer, have been associated with increased risk of metastasis and decreased overall survival. However, only few studies revealed the presence and the count of circulating epithelial cells also in peripheral blood of SCCHN patients (12-17), especially in a nonmetastatic setting. Additionally, the prognostic role of CTCs in locally advanced SCCHN has not been well studied so far. Moreover, CTCs analysis might serve as a “liquid biopsy”, since peripheral blood is much more easily accessible than tumor tissue. Therefore, the identification of molecules contributing to tumorcell invasion, metastasis or therapy resistance in CTCs would potentially allow identification of novel drug targets or modification of the assigned therapy. An example of a molecule which was proven to play a major role in cancer development and metastasis is the epidermal growth factor receptor (EGFR). EGFR is commonly expressed on the surface of normal epithelial cells and at high levels in a variety of epithelial cell-derived tumors including SCCHN (18). The aberrant activation of the EGFR in SCCHN triggers a series of intracellular signals ultimately leading to the proliferation of cancer cells, induction of angiogenesis and metastasis (19). These observations suggest a link between EGFR activation and the generation of circulating metastatic cells. The latter is initiated by a disruption of the cell-to-cell adhesion mechanisms in the epithelial cell layer and acquisition of cell mobility, which is hallmarked by the activation of a cellular program termed epithelial–mesenchymal transition (EMT). EGFR has been suggested by a number of studies as a potent major member of the EMT pathway, because its activation is able to induce EMT in cancer cells (20,21). Beside the role of EMT in tumor invasion and metastasis, the EMT phenotype has been shown to interfere with the response of SCCHN cells to antitumor treatment (22-25).
Transcriptional activation of replication proteins (i.e. pre-replication complex) can induce endoreduplica- tion 90 . Two E2F coding genes and one DPA gene are upregulated in HTT (Table 2 ). The heterodimer E2F-DP promotes the expression of S-phase genes. Additionally, the majority of the components required for DNA rep- lication are upregulated in the HTT dataset (Figs 5 and 6 ); this is in agreement with the hypertrophic phenotype observed 4 . Additionally, DPA expression levels have been also reported to correlate positively with final leaf size traits 64 . The RBR protein family is crucial and defined as a core cell cycle control by repressing G1/S phase cell cycle progression. RBR is known as a tumor suppressor and is inactivated in many human cancers 24 . Two RBR maize genes have been well characterized 66 , 67 , ZmRBR1 has a canonical function as repressor of cell cycle pro- gression while ZmRBR3 promotes the expression of the E2F/DP targets, including the MCM family, required for the initiation of DNA synthesis 67 . Our analysis showed a strong upregulation of ZmRBR4 and ZmRBR3 in both tumorcell types but no alterations in ZmRBR1/2 gene expressions (Table 2 and Fig. 4 ). ZmRBR4 has not yet been characterized but its strong expression in both tumorcell-types rather speaks for a positive role in cell cycle progression.
We present the time evolution of the system components in Figure 5.2 for t ⩽ 150. The main tumor propagates to the right side of the domain and degrades at the same time the ECM. In the invaded area, where proteolysis already diminished the tissue density to a small amount, new tumorcell clusters emerge. They take the form of peaks in the cancer cell concentrations. The number of these peaks as well as their maximal concentration vary in time. They evolve in time by moving and merging with each other. At the time instance t = 75 almost half of the domain is invaded by cancer cells. The interaction of the peaks continues as the invaded area becomes larger. No steady state is attained even in later time instances 300 ⩽ t ⩽ 500 when the full domain has been invaded. The remaining components of the system behave differently: we observe only small spikes in the uPA and plasmin densities while the more diffusive inhibitor PAI-1 exhibits a smooth density over space. The densities of the enzymes have not overshot a concentration of one throughout the simulation.