A molecularly distinct subset of glioblastoma requires serum-containing media to establish sustainable bona fide glioblastoma stem cell cultures.

Glioblastoma (GBM) is the most frequent and deadly primary malignant brain tumor. Hallmarks are extensive intra-tumor and inter-tumor heterogeneity and highly invasive growth, which provide great challenges for treatment. Efficient therapy is lacking and the majority of patients survive less than 1 year from diagnosis. GBM progression and recurrence is caused by treatment-resistant glioblastoma stem cells (GSCs). GSC cultures are considered important models in target identification and drug screening studies. The current state-of-the-art method, to isolate and maintain GSC cultures that faithfully mimic the primary tumor, is to use serum-free (SF) media conditions developed for neural stem cells (NSCs). Here we have investigated the outcome of explanting 218 consecutively collected GBM patient samples under both SF and standard, serum-containing media conditions. The frequency of maintainable SF cultures (SFCs) was most successful, but for a subgroup of GBM specimens, a viable culture could only be established in serum-containing media, called exclusive serum culture (ESC). ESCs expressed nestin and SOX2, and displayed all functional characteristics of a GSC, that is, extended proliferation, sustained self-renewal and orthotopic tumor initiation. Once adapted to the in vitro milieu they were also sustainable in SF media. Molecular analyses showed that ESCs formed a discrete group that was most related to the mesenchymal GBM subtype. This distinct subgroup of GBM that would have evaded modeling in SF conditions only provide unique cell models of GBM inter-tumor heterogeneity.

Heterogeneity in Malignant Pleural Mesothelioma.

Despite advances in malignant pleural mesothelioma therapy, life expectancy of affected patients remains short. The limited efficiency of treatment options is mainly caused by inter- and intra-tumor heterogeneity of mesotheliomas. This diversity can be observed at the morphological and molecular levels. Molecular analyses reveal a high heterogeneity (i) between patients; (ii) within different areas of a given tumor in terms of different clonal compositions; and (iii) during treatment over time. The aim of the present review is to highlight this diversity and its therapeutic implications.

One microenvironment does not fit all: heterogeneity beyond cancer cells.

Human cancers exhibit formidable molecular heterogeneity, to a large extent accounting for the incomplete and transitory efficacy of current anti-cancer therapies. However, neoplastic cells alone do not manifest the disease, but conscript a battery of non-tumor cells to enable and sustain hallmark capabilities of cancer. Escaping immunosurveillance is one of such capabilities. Tumors evolve immunosuppressive microenvironment to subvert anti-tumor immunity. In this review, we will focus on tumor-associated myeloid cells, which constitute an essential part of the immune microenvironment and reciprocally interact with cancer cells to establish malignancy toward metastasis. The diversity and plasticity of these cells constitute another layer of heterogeneity, beyond the heterogeneity of cancer cells themselves. We envision that immune microenvironment co-evolves with the genetic heterogeneity of tumor. Addressing the question of how genetically distinct tumors shape and are shaped by unique immune microenvironment will provide an attractive rationale to develop novel immunotherapeutic modalities. Here, we discuss the complex nature of tumor microenvironment, with an emphasis on the cellular and functional heterogeneity among tumor-associated myeloid cells as well as immune environment heterogeneity in the context of a full spectrum of human breast cancers.

The Vicious Cycle of Melanoma-Microglia Crosstalk: Inter-Melanoma Variations in the Brain-Metastasis-Promoting IL-6/JAK/STAT3 Signaling Pathway.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

Breast Cancer Heterogeneity.

Breast tumor heterogeneity is a major challenge in the clinical management of breast cancer patients. Both inter-tumor and intra-tumor heterogeneity imply that each breast cancer (BC) could have different prognosis and would benefit from specific therapy. Breast cancer is a dynamic entity, changing during tumor progression and metastatization and this poses fundamental issues to the feasibility of a personalized medicine approach. The most effective therapeutic strategy for patients with recurrent disease should be assessed evaluating biopsies obtained from metastatic sites. Furthermore, the tumor progression and the treatment response should be strictly followed and radiogenomics and liquid biopsy might be valuable tools to assess BC heterogeneity in a non-invasive way.

EphA2 and EGFR: Friends in Life, Partners in Crime. Can EphA2 Be a Predictive Biomarker of Response to Anti-EGFR Agents?

The Eph receptors represent the largest group among Receptor Tyrosine kinase (RTK) families. The Eph/ephrin signaling axis plays center stage during development, and the deep perturbation of signaling consequent to its dysregulation in cancer reveals the multiplicity and complexity underlying its function. In the last decades, they have emerged as key players in solid tumors, including colorectal cancer (CRC); however, what causes EphA2 to switch between tumor-suppressive and tumor-promoting function is still an active theater of investigation. This review summarizes the recent advances in understanding EphA2 function in cancer, with detail on the molecular determinants of the oncogene-tumor suppressor switch function of EphA2. We describe tumor context-specific examples of EphA2 signaling and the emerging role EphA2 plays in supporting cancer-stem-cell-like populations and overcoming therapy-induced stress. In such a frame, we detail the interaction of the EphA2 and EGFR pathway in solid tumors, including colorectal cancer. We discuss the contribution of the EphA2 oncogenic signaling to the resistance to EGFR blocking agents, including cetuximab and TKIs.

Implications of non-uniqueness in phylogenetic deconvolution of bulk DNA samples of tumors.

BACKGROUND: Tumors exhibit extensive intra-tumor heterogeneity, the presence of groups of cellular populations with distinct sets of somatic mutations. This heterogeneity is the result of an evolutionary process, described by a phylogenetic tree. In addition to enabling clinicians to devise patient-specific treatment plans, phylogenetic trees of tumors enable researchers to decipher the mechanisms of tumorigenesis and metastasis. However, the problem of reconstructing a phylogenetic tree T given bulk sequencing data from a tumor is more complicated than the classic phylogeny inference problem. Rather than observing the leaves of T directly, we are given mutation frequencies that are the result of mixtures of the leaves of T. The majority of current tumor phylogeny inference methods employ the perfect phylogeny evolutionary model. The underlying Perfect Phylogeny Mixture (PPM) combinatorial problem typically has multiple solutions. RESULTS: We prove that determining the exact number of solutions to the PPM problem is #P-complete and hard to approximate within a constant factor. Moreover, we show that sampling solutions uniformly at random is hard as well. On the positive side, we provide a polynomial-time computable upper bound on the number of solutions and introduce a simple rejection-sampling based scheme that works well for small instances. Using simulated and real data, we identify factors that contribute to and counteract non-uniqueness of solutions. In addition, we study the sampling performance of current methods, identifying significant biases. CONCLUSIONS: Awareness of non-uniqueness of solutions to the PPM problem is key to drawing accurate conclusions in downstream analyses based on tumor phylogenies. This work provides the theoretical foundations for non-uniqueness of solutions in tumor phylogeny inference from bulk DNA samples.

Involvement of Chromatin Remodeling Genes and the Rho GTPases RhoB and CDC42 in Ovarian Clear Cell Carcinoma.

OBJECTIVE: Ovarian clear cell carcinomas (OCCCs) constitute a rare ovarian cancer subtype with distinct clinical features, but may nonetheless be difficult to distinguish morphologically from other subtypes. There is limited knowledge of genetic events driving OCCC tumorigenesis beyond ARID1A, which is reportedly mutated in 30-50% of OCCCs. We aimed to further characterize OCCCs by combined global transcriptional profiling and targeted deep sequencing of a panel of well-established cancer genes. Increased knowledge of OCCC-specific genetic aberrations may help in guiding development of targeted treatments and ultimately improve patient outcome. METHODS: Gene expression profiling of formalin-fixed, paraffin-embedded (FFPE) tissue from a cohort of the major ovarian cancer subtypes (cohort 1; n = 67) was performed using whole-genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL) bead arrays, followed by pathway, gene module score, and gene ontology analyses, respectively. A second FFPE cohort of 10 primary OCCCs was analyzed by targeted DNA sequencing of a panel of 60 cancer-related genes (cohort 2). Non-synonymous and non-sense variants affecting single-nucleotide variations and insertions or deletions were further analyzed. A tissue microarray of 43 OCCCs (cohort 3) was used for validation by immunohistochemistry and chromogenic in situ hybridization. RESULTS: Gene expression analyses revealed a distinct OCCC profile compared to other histological subtypes, with, e.g., ERBB2, TFAP2A, and genes related to cytoskeletal actin regulation being overexpressed in OCCC. ERBB2 was, however, not overexpressed on the protein level and ERBB2 amplification was rare in the validation cohort. Targeted deep sequencing revealed non-synonymous variants or insertions/deletions in 11/60 cancer-related genes. Genes involved in chromatin remodeling, including ARID1A, SPOP, and KMT2D were frequently mutated across OCCC tumors. CONCLUSION: OCCCs appear genetically heterogeneous, but harbor frequent alterations in chromatin remodeling genes. Overexpression of TFAP2A and ERBB2 was observed on the mRNA level in relation to other ovarian cancer subtypes. However, overexpression of ERBB2 was not reflected by HER2 amplification or protein overexpression in the OCCC validation cohort. In addition, Rho GTPase-dependent actin organization may also play a role in OCCC pathogenesis and warrants further investigation. The distinct biological features of OCCC discovered here may provide a basis for novel targeted treatment strategies.