Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER + /PR + model (SSM2), a Her2 + model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters.

BackgroundStudies assessing immune parameters typically utilize human PBMCs or murine splenocytes to generate data that is interpreted as representative of immune status. Using splenocytes, we have shown memory CD4-T cells that expand following systemic immunostimulatory therapies undergo rapid IFNg-mediated activation induced cell death (AICD) resulting in a net loss of total CD4-T cells which correlates with elevated PD-1 expression. This is in contrast to CD8-T cells which expand with minimal PD-1 upregulation and apoptosis. In this study we expand upon our previous work by evaluating CD4 and CD8-T cell phenotype and distribution in peripheral organs which are more representative of immune responses occurring at metastatic sites following immunotherapy.MethodsPhenotypic assessment of T cells in both lymphoid (spleen and LN) as well as peripheral organs (liver and lungs) in control and immunotherapy treated mice was performed to survey the impact of location on memory phenotype and activation marker status. Peripheral blood from patients undergoing systemic high dose IL-2 was also assessed for expression of PD-1 and memory phenotype.ResultsHere we reveal that, similar to what occurs in the spleen and lymph nodes, CD4-T cell numbers decreased while CD8-T cells expanded at these peripheral sites. In contrast to having differential expression of PD-1 as occurs in the spleen, both CD4 and CD8-T cells had significantly elevated levels of PD-1 in both the liver and lungs. Further analysis correlated PD-1 expression to CD62Llow (T effector/effector memory,TE/EM) expression which are more prevalent in CD4-T cells in general as well as CD8-T cells in peripheral organs. Similar elevated PD-1 expression on TE/EM cells was observed in patients undergoing systemic high-dose IL-2 therapy.ConclusionsThese data highlight PD-1 expressing and/or TE/EM subsets of T cells in circulation as more representative of cells at immune sites and underscore the importance of valuation both in lymphoid as well as target organs when making determinations about immune status.Trial registrationClinicalTrials.gov NCT01416831. Registered August 12, 2011.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-017-0235-4) contains supplementary material, which is available to authorized users.

Background:The integrin binding-defective mutant of IGF1 (R36E/R37E) is functionally defective and does not induce ternary complex formation (integrin-IGF1-IGF1R). Results: R36E/R37E suppressed signaling induced by WT IGF1, the binding of WT IGF1 to cells, ternary complex formation, cell viability, and tumorigenesis. Conclusion: R36E/R37E is a dominant-negative antagonist of IGF signaling. Significance: R36E/R37E has potential as a therapeutic agent.

Purpose: We apply positron emission tomography (PET) to elucidate changes in nanocarrier extravasation during the transition from premalignant to malignant cancer, providing insight into the use of imaging to characterize early cancerous lesions and the utility of nanoparticles in early disease.Experimental Design: Albumin and liposomes were labeled with 64 Cu (half-life 12.7 hours), and longitudinal PET and CT imaging studies were conducted in a mouse model of ductal carcinoma in situ. A pharmacokinetic model was applied to estimate the tumor vascular volume and permeability.Results: From early time points characterized by disseminated hyperproliferation, the enhanced vascular permeability facilitated lesion detection. During disease progression, the vascular volume fraction increased 1.6-fold and the apparent vascular permeability to albumin and liposomes increased $2.5-fold to 6.6 Â 10 À8 and 1.3 Â 10 À8 cm/s, respectively, with the accumulation of albumin increasing earlier in the disease process. In the malignant tumor, both tracers reached similar mean intratumoral concentrations of $6% ID/cc but the distribution of liposomes was more heterogeneous, ranging from 1% to 18% ID/cc compared with 1% to 9% ID/cc for albumin. The tumor-to-muscle ratio was 17.9 AE 8.1 and 7.1 AE 0.5 for liposomes and albumin, respectively, indicating a more specific delivery of liposomes than with albumin. Conclusions: PET imaging of radiolabeled particles, validated by confocal imaging and histology, detected the transition from premalignant to malignant lesions and effectively quantified the associated changes in vascular permeability.