Significance Using microfiltration as a liquid biopsy for the recovery of circulating tumor cells (CTCs) has revealed an accompanying macrophage subset that we use as a highly sensitive biomarker for solid tumors. We supply evidence that this circulating giant cell is a subset of disseminated tumor-associated macrophages capable of binding CTCs in peripheral blood of cancer patients. The presence of this cell expands the concept of using a liquid biopsy not only to indicate cancer presence but also to track cancer treatment effects sequentially using other circulating blood cells. Further, we supply observational evidence hypothesizing a metastasis pathway model in which CTCs migrate with pro-angiogenic macrophages, linking cancer cell intravasation, migration, and extravasation and the formation of metastatic microenvironments.

Desmoplasia, a fibrotic mass including cancer-associated fibroblasts (CAFs) and self-sustaining extracellular matrix (D-ECM), is a puzzling feature of pancreatic ductal adenocarcinoma (PDACs). Conflicting studies have identified tumor-restricting and tumor-promoting roles of PDAC-associated desmoplasia, suggesting that individual CAF/D-ECM protein constituents have distinguishable tumorigenic and tumor-repressive functions. Using 3D culture of normal pancreatic versus PDAC-associated human fibroblasts, we identified a CAF/D-ECM phenotype that correlates with improved patient outcomes, and that includes CAFs enriched in plasma membrane-localized, active α5β1-integrin. Mechanistically, we established that TGFβ is required for D-ECM production but dispensable for D-ECM-induced naïve fibroblast-to-CAF activation, which depends on αvβ5-integrin redistribution of pFAK-independent active α5β1-integrin to assorted endosomes. Importantly, the development of a simultaneous multi-channel immunofluorescence approach and new algorithms for computational batch-analysis and their application to a human PDAC panel, indicated that stromal localization and levels of active SMAD2/3 and α5β1-integrin distinguish patient-protective from patient-detrimental desmoplasia and foretell tumor recurrences, suggesting a useful new prognostic tool.DOI: http://dx.doi.org/10.7554/eLife.20600.001

Recent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch V R CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve TM ) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)1/CD452/DAPI1 cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK1/CD452 cells than CellSearch. Furthermore, analysis showed low correlation between the total CK1/CD452 cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK1/CD452/DAPI1 cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using nonEpCAM dependent isolation.

Pancreatic ductal adenocarcinoma (PDAC) carries a dismal prognosis and lacks a human cell model of early disease progression. When human PDAC cells are injected into immunodeficient mice, they generate advanced stage cancer. We hypothesized that if human PDAC cells were converted to pluripotency and then allowed to differentiate back into pancreatic tissue, they might undergo early stages of cancer. Although most induced pluripotent stem (iPS) cell lines were not of the expected cancer genotype, one PDAC line, 10-22 cells, when injected into immunodeficient mice, generates pancreatic intraepithelial neoplasia (PanIN) precursors to PDAC that progress to the invasive stage. The PanIN-like cells secrete or release proteins corresponding to genes and networks expressed in human pancreatic cancer progression and which predicted an HNF4α network also seen a mouse model. Thus, rare events allow iPS technology to provide a live human cell model of early pancreatic cancer and new insights into disease progression.

Human NK cells lyse Ab-coated target cells through the process of Ab-dependent cellular cytotoxicity (ADCC). Improving ADCC responses is desirable because it is thought to be an important antitumor mechanism for some Abs. NK cell inhibitory receptors, such as killer cell Ig-like receptors, engage with MHC class I molecules on self-cells to block NK cell activation. Accordingly, we enhanced ADCC responses by blocking NK cell inhibitory receptors, thus perturbing induction of the self-recognition signal. In a cell line model of anti-lymphoma therapy, the combination of rituximab with an Ab that blocks inhibitory self-recognition yielded increased NK cell-mediated target cell lysis when compared with rituximab alone. To validate this proof-of-concept, we then used a more representative approach in which an individual’s fresh primary NK cells encountered autologous, EBV-transformed B cells. In this system, rituximab and a combination of Abs that block NK cell inhibitory receptors yielded improved NK cell-mediated lysis over rituximab alone. The results show, for the first time, that disruption of inhibitory self-recognition can efficiently promote ADCC in a human model, applying an autologous system in which physiologic checkpoints are in place. This method provides an alternative approach to potentiate the therapeutic benefit of antitumor Abs that mediate ADCC.

Fibroblast activation protein is highly expressed in pancreatic adenocarcinoma, with greatest expression immediately adjacent to tumor. Higher FAP expression is associated with worse clinical outcome. The investigation of FAP inhibitors as a therapeutic strategy against pancreatic cancer should be considered.

No abstract

Purpose Patients with persistent/recurrent epithelial ovarian cancer/primary peritoneal cancer (EOC/PPC) have limited treatment options. AKT and PI3K pathway activation is common in EOC/PPC, resulting in constitutive activation of downstream mTOR. The GOG conducted a phase II evaluation of efficacy and safety for the mTOR inhibitor, temsirolimus in EOC/PPC and explored circulating tumor cells (CTC) and AKT/mTOR/downstream tumor markers. Methods Eligible women with measurable, persistent/recurrent EOC/PPC who had received 1–3 prior regimens were treated with 25 mg weekly IV temsirolimus until progression or intolerable toxicity. Primary endpoints were progression-free survival (PFS) ≥6-months, tumor response, and toxicity. CellSearch® system was used to examine CTC, and AKT/mTOR/downstream markers were evaluated by archival tumor immunohistochemistry. Kendall’s tau-b correlation coefficient (r) and Cox regression modeling were used to explore marker associations with baseline characteristics and outcome. Results Sixty patients were enrolled in a two-stage sequential design. Of 54 eligible and evaluable patients, 24.1% (90%CI 14.9%–38.6%) had PFS ≥6 months (median 3.1 months), 9.3% (90%CI 3.7%–23.4%) experienced a partial response. Grade 3/4 adverse events included metabolic(8), gastrointestinal(8), pain(6), constitutional(5) and pulmonary(4). Suggested associations were between cyclin D1 and PFS ≥6 months, PFS or survival; positive CTC pre-treatment and lack of response; and high CTC expression of M30 and PFS ≥6 months/longer PFS. Conclusions Temsirolimus appears to have modest activity in persistent/recurrent EOC/PPC; however, PFS is just below that required to warrant inclusion in phase III studies in unselected patients. Cyclin D1 as a selection marker and CTC measures merit further study.