Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence.

Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment

Introduction of Zinc-salt Fixation for Effective Detection of Immune Cell–related Markers by Immunohistochemistry

Tissue localization of immune cells is critical to the study of disease processes in mouse models of human diseases. However, immunohistochemistry (IHC) for immune cell phenotyping in mouse tissue sections presents specific technical challenges. For example, CD4 and CD8 have been difficult to detect using IHC on formalin-fixed and paraffin-embedded (FFPE) mouse tissue, prompting alternative methods. We investigated the use of formalin free zinc-salt fixation (ZN) (Beckstead, 1994) and optimized IHC protocols for detecting a panel of immune cell-related markers (CD3, CD4, CD8, Foxp3, B220, F4/80, CD68, MHC class-I, MHC class-II and Gr-1). The IHC results for these markers were compared on mouse spleen tissue treated with: neutral buffered formalin (NBF) or ZN with or Zn without antigen retrieval (AR). Whereas CD4 and CD8 were not detected in NBF treated tissue, all markers were detected in ZN treated tissue without AR. Thus, the use of ZN treatment for IHC staining can be a good tool for studying immuno-reactive lesions in tissues

Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence.

Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment

Pathobiology of the 129:Stat1 -/- mouse model of human age-related ER-positive breast cancer with an immune infiltrate-excluded phenotype.

BackgroundStat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds) develop estrogen receptor-positive (ER+), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1 -/- host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors.MethodsMice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129:Stat1 -/--derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells.ResultsTumorigenesis in 129:Stat1 -/- originates from a population of FoxA1+ large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1 -/- tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor.Conclusions129:Stat1 -/- is a unique model for studying the critical origins and risk reduction strategies in age-related ER+ breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies

Pathobiology of the 129:Stat1 −/− mouse model of human age-related ER-positive breast cancer with an immune infiltrate-excluded phenotype

Abstract Background Stat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds) develop estrogen receptor-positive (ER+), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1 −/− host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors. Methods Mice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129:Stat1 −/−-derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells. Results Tumorigenesis in 129:Stat1 −/− originates from a population of FoxA1+ large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1 −/− tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor. Conclusions 129:Stat1 −/− is a unique model for studying the critical origins and risk reduction strategies in age-related ER+ breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies

Additional file 1: Tables S1. of Pathobiology of the 129:Stat1 −/− mouse model of human age-related ER-positive breast cancer with an immune infiltrate-excluded phenotype

Tumor Topography and S2. Tumor incidence in 129: Stat1 -/- female mice. (PDF 90 kb

Additional file 2: Figures S1. of Pathobiology of the 129:Stat1 −/− mouse model of human age-related ER-positive breast cancer with an immune infiltrate-excluded phenotype

Milky ducts in 129: Stat1 -/- mammary gland from nulliparous mouse. Figure S2. The histological types of MIN. Figure S3. LOP cells in 129: Stat1 -/- mammary gland. Figure S4. Keratin expression in MIN. Figure S5. FoxA1 positive cells in 129: WT and 129: Stat1 -/- mice. Figure S6. FoxA1+/ER+/PR+ positive cells in Stat1-null tumor. Figure S7. Invasion and metastasis of 129: Stat1 -/- tumor. Figure S8. Stat1-null mammary fat pads poorly support Stat1-null tumor growth. Figure S9. Immune cells and type IV collagen rich microenvironment in Stat1KO tumor. (PDF 6646 kb