Increased complexity in carcinomas: Analyzing and modeling the interaction of human cancer cells with their microenvironment.

Solid cancers are not simple accumulations of malignant tumor cells but rather represent complex organ-like structures. Despite a more chaotic general appearance as compared to the highly organized setup of healthy tissues, cancers still show highly differentiated structures and a close interaction with and dependency on the interwoven connective tissue. This complexity within cancers is not known in detail at the molecular level so far. The first part of this article will shortly describe the technology and strategies to quantify and dissect the heterogeneity in human solid cancers. Moreover, there is urgent need to better understand human cancer biology since the development of novel anti-cancer drugs is far from being efficient, predominantly due to the scarcity of predictive preclinical models. Hence, in vivo and in vitro models were developed, which better recapitulate the complexity of human cancers, by their intrinsic three-dimensional nature and the cellular heterogeneity and allow functional intervention for hypothesis testing. Therefore, in the second part 3D in vitro cancer models are presented that analyze and depict the heterogeneity in human cancers. Advantages and drawbacks of each model are highlighted and their suitability to preclinical drug testing is discussed.

Recruitment of Scc2/4 to double-strand breaks depends on γH2A and DNA end resection.

Homologous recombination enables cells to overcome the threat of DNA double-strand breaks (DSBs), allowing for repair without the loss of genetic information. Central to the homologous recombination repair process is the de novo loading of cohesin around a DSB by its loader complex Scc2/4. Although cohesin's DSB accumulation has been explored in numerous studies, the prerequisites for Scc2/4 recruitment during the repair process are still elusive. To address this question, we combine chromatin immunoprecipitation-qPCR with a site-specific DSB in vivo, in Saccharomyces cerevisiae We find that Scc2 DSB recruitment relies on γH2A and Tel1, but as opposed to cohesin, not on Mec1. We further show that the binding of Scc2, which emanates from the break site, depends on and coincides with DNA end resection. Absence of chromatin remodeling at the DSB affects Scc2 binding and DNA end resection to a comparable degree, further indicating the latter to be a major driver for Scc2 recruitment. Our results shed light on the intricate DSB repair cascade leading to the recruitment of Scc2/4 and subsequent loading of cohesin.

Therapeutic paradigm of dual targeting VEGF and PDGF for effectively treating FGF-2 off-target tumors.

FGF-2 displays multifarious functions in regulation of angiogenesis and vascular remodeling. However, effective drugs for treating FGF-2+ tumors are unavailable. Here we show that FGF-2 modulates tumor vessels by recruiting NG2+ pricytes onto tumor microvessels through a PDGFRß-dependent mechanism. FGF-2+ tumors are intrinsically resistant to clinically available drugs targeting VEGF and PDGF. Surprisingly, dual targeting the VEGF and PDGF signaling produces a superior antitumor effect in FGF-2+ breast cancer and fibrosarcoma models. Mechanistically, inhibition of PDGFRß ablates FGF-2-recruited perivascular coverage, exposing anti-VEGF agents to inhibit vascular sprouting. These findings show that the off-target FGF-2 is a resistant biomarker for anti-VEGF and anti-PDGF monotherapy, but a highly beneficial marker for combination therapy. Our data shed light on mechanistic interactions between various angiogenic and remodeling factors in tumor neovascularization. Optimization of antiangiogenic drugs with different principles could produce therapeutic benefits for treating their resistant off-target cancers.

Dual roles of endothelial FGF-2-FGFR1-PDGF-BB and perivascular FGF-2-FGFR2-PDGFRß signaling pathways in tumor vascular remodeling.

Perivascular cells are important cellular components in the tumor microenvironment (TME) and they modulate vascular integrity, remodeling, stability, and functions. Here we show using mice models that FGF-2 is a potent pericyte-stimulating factor in tumors. Mechanistically, FGF-2 binds to FGFR2 to stimulate pericyte proliferation and orchestrates the PDGFRß signaling for vascular recruitment. FGF-2 sensitizes the PDGFRß signaling through increasing PDGFRß levels in pericytes. To ensure activation of PDGFRß, the FGF-2-FGFR1-siganling induces PDGF-BB and PDGF-DD, two ligands for PDGFRß, in angiogenic endothelial cells. Thus, FGF-2 directly and indirectly stimulates pericyte proliferation and recruitment by modulating the PDGF-PDGFRß signaling. Our study identifies a novel mechanism by which the FGF-2 and PDGF-BB collaboratively modulate perivascular cell coverage in tumor vessels, thus providing mechanistic insights of pericyte-endothelial cell interactions in TME and conceptual implications for treatment of cancers and other diseases by targeting the FGF-2-FGFR-pericyte axis.

Exclusion from spheroid formation identifies loss of essential cell-cell adhesion molecules in colon cancer cells.

Many cell lines derived from solid cancers can form spheroids, which recapitulate tumor cell clusters and are more representative of the in vivo situation than 2D cultures. During spheroid formation, a small proportion of a variety of different colon cancer cell lines did not integrate into the sphere and lost cell-cell adhesion properties. An enrichment protocol was developed to augment the proportion of these cells to 100% purity. The basis for the separation of spheroids from non-spheroid forming (NSF) cells is simple gravity-sedimentation. This protocol gives rise to sub-populations of colon cancer cells with stable loss of cell-cell adhesion. SW620 cells lacked E-cadherin, DLD-1 cells lost α-catenin and HCT116 cells lacked P-cadherin in the NSF state. Knockdown of these molecules in the corresponding spheroid-forming cells demonstrated that loss of the respective proteins were indeed responsible for the NSF phenotypes. Loss of the spheroid forming phenotype was associated with increased migration and invasion properties in all cell lines tested. Hence, we identified critical molecules involved in spheroid formation in different cancer cell lines. We present here a simple, powerful and broadly applicable method to generate new sublines of tumor cell lines to study loss of cell-cell adhesion in cancer progression.

Modeling human carcinomas: physiologically relevant 3D models to improve anti-cancer drug development.

Anti-cancer drug development is inefficient, mostly due to lack of efficacy in human patients. The high fail rate is partly due to the lack of predictive models or the inadequate use of existing preclinical test systems. However, progress has been made and preclinical models were improved or newly developed, which all account for basic features of solid cancers, three-dimensionality and heterotypic cell interaction. Here we give an overview of available in vivo and in vitro models of cancer, which meet the criteria of being 3D and mirroring human tumor-stroma interactions. We only focus on drug response models without touching models for pharmacokinetic and dynamic, toxicity or delivery aspects.

The Resazurin Reduction Assay Can Distinguish Cytotoxic from Cytostatic Compounds in Spheroid Screening Assays.

Spheroid-based cellular screening approaches represent a highly physiologic experimental setup to identify novel anticancer drugs and an innovative preclinical model to reduce the high failure rate of anticancer compounds in clinical trials. The resazurin reduction (RR) assay, known as the alamarBlue or CellTiter-Blue assay, is frequently used to determine cell viability/proliferation capacity in eukaryotic cells. Whether this assay is applicable to assess viability in multicellular spheroids has not been evaluated. We analyzed the RR assay to measure cytotoxic and/or cytostatic responses in tumor cell spheroids compared with conventional 2D cultures. We found that tight cell-cell interactions in compact spheroids hamper resazurin uptake and its subsequent reduction to resorufin, leading to lowered reduction activity in relation to the actual cellular health/cell number. Treatment with staurosporine disrupted close cell-cell contacts, which increased resazurin reduction compared with untreated controls. Loss of tight junctions by trypsinization or addition of EGTA or EDTA restored high resazurin reduction rates in untreated spheroids. In conclusion, the RR assay is unsuited to quantitatively measure cellular health/cell number in compact spheroids. However, it can be used to distinguish between cytotoxic versus cytostatic compounds in spheroids. Restoration of the correlation of cell viability/number to resazurin reduction capacity can be achieved by disruption of tight junctions.