ABSTRACT
T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.
Subject(s)
High-Throughput Nucleotide Sequencing , T-Lymphocytes , Base Sequence , Chromosome Mapping , High-Throughput Nucleotide Sequencing/methods , Receptors, Antigen, T-Cell, alpha-beta/geneticsABSTRACT
T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.
ABSTRACT
Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.
Subject(s)
Breast Neoplasms , Biopsy , Breast Neoplasms/genetics , Female , Humans , Tumor Microenvironment/geneticsABSTRACT
Complement is a critical component of humoral immunity implicated in cancer development; however, its biological contributions to tumorigenesis remain poorly understood. Using the K14-HPV16 transgenic mouse model of squamous carcinogenesis, we report that urokinase (uPA)+ macrophages regulate C3-independent release of C5a during premalignant progression, which in turn regulates protumorigenic properties of C5aR1+ mast cells and macrophages, including suppression of CD8+ T cell cytotoxicity. Therapeutic inhibition of C5aR1 via the peptide antagonist PMX-53 improved efficacy of paclitaxel chemotherapy associated with increased presence and cytotoxic properties of CXCR3+ effector memory CD8+ T cells in carcinomas, dependent on both macrophage transcriptional programming and IFNγ. Together, these data identify C5aR1-dependent signaling as an important immunomodulatory program in neoplastic tissue tractable for combinatorial cancer immunotherapy.
Subject(s)
Carcinogenesis/drug effects , Complement C5a/drug effects , Drug Therapy , Receptor, Anaphylatoxin C5a/drug effects , Animals , CD8-Positive T-Lymphocytes/drug effects , Carcinoma, Squamous Cell/drug therapy , Disease Models, Animal , Drug Therapy/methods , Humans , Macrophages/drug effects , Macrophages/physiology , Mice , Signal Transduction/drug effectsABSTRACT
Here, we describe a multiplexed immunohistochemical platform with computational image processing workflows, including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination and revealed that response to therapy correlated with degree of mono-myelocytic cell density and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification and provide digital image processing pipelines to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to improve biomarker discovery and assessment.
