Quantitative, Architectural Analysis of Immune Cell Subsets in Tumor-Draining Lymph Nodes from Breast Cancer Patients and Healthy Lymph Nodes

Background: To date, pathological examination of specimens remains largely qualitative. Quantitative measures of tissue spatial features are generally not captured. To gain additional mechanistic and prognostic insights, a need for quantitative architectural analysis arises in studying immune cell-cancer interactions within the tumor microenvironment and tumor-draining lymph nodes (TDLNs). Methodology/Principal Findings: We present a novel, quantitative image analysis approach incorporating 1) multi-color tissue staining, 2) high-resolution, automated whole-section imaging, 3) custom image analysis software that identifies cell types and locations, and 4) spatial statistical analysis. As a proof of concept, we applied this approach to study the architectural patterns of T and B cells within tumor-draining lymph nodes from breast cancer patients versus healthy lymph nodes. We found that the spatial grouping patterns of T and B cells differed between healthy and breast cancer lymph nodes, and this could be attributed to the lack of B cell localization in the extrafollicular region of the TDLNs. Conclusions/Significance: Our integrative approach has made quantitative analysis of complex visual data possible. Our results highlight spatial alterations of immune cells within lymph nodes from breast cancer patients as an independent variable from numerical changes. This opens up new areas of investigations in research and medicine. Future application of this approach will lead to a better understanding of immune changes in the tumor microenvironment and TDLNs, and how they affect clinical outcome

Supplementary Figure Legend from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas

Supplementary Figure Legend from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas</jats:p

Supplementary Figure Legend from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas

Supplementary Figure Legend from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas</jats:p

Supplementary Figure 1 from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas

Supplementary Figure 1 from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas</jats:p

Supplementary Figure 1 from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas

Supplementary Figure 1 from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas</jats:p

Data from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas

&lt;div&gt;Abstract&lt;p&gt;Expression of transporter associated with antigen processing (TAP) is often lost in metastatic carcinomas, resulting in defective antigen processing and presentation and escape of the cancer cells from immune surveillance. In this study, the nature of TAP deficiencies in tumors was investigated. By chromatin immunoprecipitation assay, we showed that the recruitment of RNA polymerase II to the &lt;i&gt;TAP-1&lt;/i&gt; gene was impaired in TAP-deficient cells derived from murine melanoma, prostate, and lung carcinomas, compared with TAP-expressing fibroblasts and lymphoma cells. This suggested that the deficiency in TAP-1 expression resulted, at least partially, from a relatively low level of transcription of the &lt;i&gt;TAP-1&lt;/i&gt; gene. Furthermore, levels of TAP-1 promoter activity, as assessed by stable transfections with a reporter construct containing the TAP-1 promoter, were relatively low in TAP-deficient cells. To examine genetic heritability of regulators of TAP-1 promoter activity, TAP- and MHC class I–deficient cells of H-2&lt;sup&gt;b&lt;/sup&gt; origin were fused with wild-type fibroblasts of H-2&lt;sup&gt;k&lt;/sup&gt; origin. Fusion with TAP-expressing cells complemented the low levels of TAP-1 promoter activity in TAP-deficient cells. However, these fused cells exhibited lower levels of TAP-1 mRNA and H-2&lt;sup&gt;k&lt;/sup&gt; than unfused fibroblasts. Further analysis showed that TAP-1 mRNA stability was lower in fused carcinoma fibroblasts than in unfused fibroblasts. Based on these results, we propose that TAP deficiency in many carcinomas is caused by a decrease in activity/expression of &lt;i&gt;trans&lt;/i&gt;-acting factors regulating TAP-1 promoter activity, as well as a decrease in TAP-1 mRNA stability. These results have significant implications for understanding immune evasion mechanisms in tumors.&lt;/p&gt;&lt;/div&gt;</jats:p

Data from Identification of Mechanisms Underlying Transporter Associated with Antigen Processing Deficiency in Metastatic Murine Carcinomas

&lt;div&gt;Abstract&lt;p&gt;Expression of transporter associated with antigen processing (TAP) is often lost in metastatic carcinomas, resulting in defective antigen processing and presentation and escape of the cancer cells from immune surveillance. In this study, the nature of TAP deficiencies in tumors was investigated. By chromatin immunoprecipitation assay, we showed that the recruitment of RNA polymerase II to the &lt;i&gt;TAP-1&lt;/i&gt; gene was impaired in TAP-deficient cells derived from murine melanoma, prostate, and lung carcinomas, compared with TAP-expressing fibroblasts and lymphoma cells. This suggested that the deficiency in TAP-1 expression resulted, at least partially, from a relatively low level of transcription of the &lt;i&gt;TAP-1&lt;/i&gt; gene. Furthermore, levels of TAP-1 promoter activity, as assessed by stable transfections with a reporter construct containing the TAP-1 promoter, were relatively low in TAP-deficient cells. To examine genetic heritability of regulators of TAP-1 promoter activity, TAP- and MHC class I–deficient cells of H-2&lt;sup&gt;b&lt;/sup&gt; origin were fused with wild-type fibroblasts of H-2&lt;sup&gt;k&lt;/sup&gt; origin. Fusion with TAP-expressing cells complemented the low levels of TAP-1 promoter activity in TAP-deficient cells. However, these fused cells exhibited lower levels of TAP-1 mRNA and H-2&lt;sup&gt;k&lt;/sup&gt; than unfused fibroblasts. Further analysis showed that TAP-1 mRNA stability was lower in fused carcinoma fibroblasts than in unfused fibroblasts. Based on these results, we propose that TAP deficiency in many carcinomas is caused by a decrease in activity/expression of &lt;i&gt;trans&lt;/i&gt;-acting factors regulating TAP-1 promoter activity, as well as a decrease in TAP-1 mRNA stability. These results have significant implications for understanding immune evasion mechanisms in tumors.&lt;/p&gt;&lt;/div&gt;</jats:p