A high throughput histology (microTMA) platform was applied for testing drugs against tumors in a novel 3D heterotypic glioblastoma brain sphere (gBS) model consisting of glioblastoma tumor cells, iPSC-derived neurons, glial cells and astrocytes grown in a spheroid. The differential responses of gBS tumors and normal neuronal cells to sustained treatments with anti-cancer drugs temozolomide (TMZ) and doxorubicin (DOX) were investigated. gBS were exposed to TMZ or DOX over a 7-day period. Untreated gBS tumors increased in size over a 4-week culture period, however, there was no increase in the number of normal neuronal cells. TMZ (100 uM) and DOX (0.3 uM) treatments caused ~30% (P~0.07) and ~80% (P < 0.001) decreases in the size of the tumors, respectively. Neither treatment altered the number of normal neuronal cells in the model. The anti-tumor effects of TMZ and DOX were mediated in part by selective induction of apoptosis. This platform provides a novel approach for screening new anti-glioblastoma agents and evaluating different treatment options for a given patient.

Background:Histopathological prognostication relies on morphological pattern recognition, but as numbers of biomarkers increase, human prognostic pattern-recognition ability decreases. Follicular lymphoma (FL) has a variable outcome, partly determined by FOXP3 Tregs. We have developed an automated method, hypothesised interaction distribution (HID) analysis, to analyse spatial patterns of multiple biomarkers which we have applied to tumour-infiltrating lymphocytes in FL.Methods:A tissue microarray of 40 patient samples was used in triplex immunohistochemistry for FOXP3, CD3 and CD69, and multispectral imaging used to determine the numbers and locations of CD3+, FOXP3/CD3+ and CD69/CD3+ T cells. HID analysis was used to identify associations between cellular pattern and outcome.Results:Higher numbers of CD3+ (P=0.0001), FOXP3/CD3+ (P=0.0031) and CD69/CD3+ (P=0.0006) cells were favourable. Cross-validated HID analysis of cell pattern identified patient subgroups with statistically significantly different survival (35.5 vs 142 months, P=0.00255), a more diffuse pattern associated with favourable outcome and an aggregated pattern with unfavourable outcome.Conclusions:A diffuse pattern of FOXP3 and CD69 positivity was favourable, demonstrating ability of HID analysis to automatically identify prognostic cellular patterns. It is applicable to large numbers of biomarkers, representing an unsupervised, automated method for identification of undiscovered prognostic cellular patterns in cancer tissue samples.

The nature of the cellular infiltration and the distribution of Treponema pallidum during rabbit testicular infection were examined by immunofluorescence and light microscopy. Low numbers of treponemes are demonstrable in the perivascular regions on day 3 post-infection. On days 6, 10, and 13, large numbers of organisms are found in the interstitial spaces. The treponemes do not appear to invade the walls or lumina of the seminiferous tubules, although tubular atrophy is obvious. On days 17, 24 and 31, treponemes are no longer identifiable by immunofluorescence in infected testicles. The cellular infiltration, which is apparent on day 6, reaches its peak on day 13, corresponding to the amount of swelling observed grossly. The infiltrate is primarily lymphocytic, but macrophages are also observed during the peak cellular response. These cells are located, as are the treponemes, in the interstitial spaces. The lymphocytes are demonstrated by specific immunofluorescence to be predominantly T cells. The peak T cell infiltration in the infected testicle is followed rapidly by the disappearance of the organisms from that organ. Thus, it is postulated that infiltration by specifically sensitized T cells results in the clearance of large numbers of T. pallidum from infected tissues.

Background. Tumor-infiltrating lymphocytes (TILs) are present in the tumor microenvironment of many cancers, with consequent tumor immunogenicity and association with survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. However, given the complexity of TIL phenotype their visualization in situ is difficult, and impossible without multiplex immunophenotyping. An understanding of both the phenotype and spatial distribution of TILs in situ within the tumor microenvironment would be advantageous to understanding their role in tumour immunobiology, especially given growing interest in tumour immunotherapy. Here we present a multi-marker, computer-aided method for analysing the distributions of CD3/FOXP3 (Treg) and CD3/CD69 (Tact) T cells in follicular lymphoma sections using a multispectral imaging (MSI) and automated analysis approach. An hypothesized interaction distance (HID) analysis was used to determine whether the spatial patterns of Tregs and Tacts were prognostically significant Design. A single section of a tissue microarray containing triplex follicular lymphoma cores from 40 subjects [24 male, 16 female, age 35 to 75 years at diagnosis, median 55 years , 2- 171 months follow-up] was stained for CD3, FOXP3, CD69 and hematoxylin. Each core was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. CD3+ TILs were located using automated image analysis. The FOXP3 and CD69 status of each CD3+ TIL was then determined and the spatial distributions of the CD3/FOXP3 and CD3/CD69 cells were used as input into the HID analysis. Results. Multiplexed IHC staining, MSI and automated per-cell quantitative analysis was successful. Kaplan-Meier analysis demonstrated favourable outcome with higher numbers of CD3+, CD3+/FOXP3+ and CD3+/CD69+ cells. HID analysis demonstrated the association of favourable outcome with a high entropy , representative of a diffuse spatial pattern, of FOXP3+ and CD69+ positive T cells. Conclusion. In this study we report that higher Treg cell counts in a diffuse pattern was associated with favorable prognosis. This supports the importance of Tregs in the tumour microenvironment. It is pertinent to mention that contradictory findings are routinely reported from studies investigating the role of Tregs in solid and haematological malignancies. This is due to the complex interactions between pro-/anti- tumour immune factors present in the tumour microenvironment. The resultant effects are due to the summation of the activities of these factors. It is therefore even more relevant that a method such as exhibited here, capable of defining and measuring the effect on patient outcome of the spatial patterns of multiple cellular phenotypes in the tumor microenvironment is available. Citation Format: Lilli Nelson, James Mansfield, Roslyn Lloyd, Chris van der Loos, Chris Rose, Richard Byers. Investigation of the spatial heterogeneity of specific immune cell phenotypes in the tumor microenvironment of follicular lymphoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A24. doi:10.1158/1538-7445.CHTME14-A24

Background. In many cancers, tumor-infiltrating lymphocytes (TILs) indicate levels of tumor immunogenicity and are a strong predictor of survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. However, visual TIL assessment cannot easily determine the phenotype of lymphocyte in situ. An understanding of both the phenotype and spatial distribution of TILs in situ within tumor regions would be advantageous. Here we present a multi-marker, computer-aided prognostication method for analysing the distributions of CD3/FOXP3 (Treg) and CD3/CD69 (Tact) T cells in follicular lymphoma sections using a multispectral imaging (MSI) and automated analysis approach. An hypothesized interaction distance (HID) analysis was used to determine whether the spatial patterns of Tregs and Tacts was prognostically significant. Design. A single section of a tissue microarray containing triplex follicular lymphoma cores from 40 subjects [24 male, 16 female, age 35 to 75 years at diagnosis, median 55 years , 2- 171 months follow-up] was stained for CD3, FOXP3, CD69 and hematoxylin. Each core was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. CD3+ TILs were located using automated image analysis. The FOXP3 and CD69 status of each CD3+ TIL was then determined and the spatial distributions of the CD3/FOXP3 and CD3/CD69 cells were used as input into the HID analysis. Results. Multiplexed IHC staining, MSI and automated per-cell quantitative analysis was successful. Kaplan-Meier analysis demonstrated favorable outcome with higher numbers of CD3+, CD3+/FOXP3+ and CD3+/CD69+ cells. HID analysis demonstrated the association of favorable outcome with a high entropy / diffuse pattern of FOXP3+ and CD69+ positive T cells. Conclusion. In this study we report that higher Treg cell counts in a diffuse pattern was associated with favorable prognosis. This supports the importance of Tregs in the tumor microenvironment. It is pertinent to mention that contradictory findings are routinely reported from studies investigating the role of Tregs in solid and haematological malignancies. This is due to the complex interactions between pro-/anti- tumor immune factors present in the tumor microenvironment. The resultant effects are due to the summation of the activities of these factors. It is therefore even more relevant that a method such as exhibited here, capable of defining, and measuring the effect on biological behaviour, in this case patient outcome, of cellular pattern is available, as demonstrated for the HID method in the present study. Citation Format: James Mansfield, Lilli Nelson, Roslyn Lloyd, Chris van der Loos, Ken Oguejiofor, Lia Menasce, Kim Linton, Chris Rose, Richard J. Byers. Favorable diffuse prognostic pattern of FOXP3+ and CD69+ T cells in follicular lymphoma demonstrated using automated imaging and analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1647. doi:10.1158/1538-7445.AM2014-1647