Differential expression of small bowel TGFβ1 and TGFβ3 characterizes intestinal strictures in patients with fibrostenotic Crohn's disease.

Small bowel strictures remain a debilitating consequence of Crohn's disease and contribute to poor outcomes for patients. Recently, TGFβ has been identified as an important driver of intestinal fibrosis. We studied the localization of TGFβ isoforms in ileal strictures of patients with Crohn's disease using in situ hybridization to understand TGFβ's role in stricture formation.

Mass Spectrometry-Based Tissue Imaging of the Tumor Microenvironment.

Multiplex ion beam imaging (MIBI) and imaging mass cytometry (IMC) enable highly multiplexed antibody (40+) staining of frozen or formalin fixed, paraffin-embedded (FFPE) human or murine tissues through detection of metal ions liberated from primary antibodies by time-of-flight mass spectrometry (TOF). These methods make detection of more than 50 targets theoretically possible while maintaining spatial orientation. As such, they are ideal tools to identify the multiple immune, epithelial, and stromal cell subsets in the tumor microenvironment and to characterize spatial relationships and tumor-immune status in either murine models or human samples.

Preclinical development of ZED8, an Zr immuno-PET reagent for monitoring tumor CD8 status in patients undergoing cancer immunotherapy.

Background: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1].

Methods: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function.

Development of T-cell engagers selective for cells co-expressing two antigens.

T cell-engaging bispecific antibodies (TCEs) are clinically effective treatments for hematological cancers. While the utility of TCEs in solid malignancies is being explored, toxicities arising from antigen expression on normal tissues have slowed or halted several clinical trials. Here, we describe the development of TCEs that preferentially drive T cell-mediated death against target cells co-expressing two tumor-associated antigens.

LRRC15 myofibroblasts dictate the stromal setpoint to suppress tumour immunity.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15). However, the molecular signals that underlie the development of LRRC15 cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFβ receptor type 2 signalling in healthy dermatopontin universal fibroblasts is essential for the development of cancer-associated LRRC15 myofibroblasts.

Activation-Induced Cytidine Deaminase Impacts the Primary Antibody Repertoire in Naive Mice.

Genetic and environmental cues shape the evolution of the B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating Ig diversity through isotype class switching and somatic mutations, which then directly influence clonal selection. Impaired B cell development in AID-knockout mice has made it difficult to study Ig diversification in an aging repertoire.

Immune contexture of paediatric cancers.

Background: The clinical development of immune checkpoint-targeted immunotherapies has been disappointing so far in paediatric solid tumours. However, as opposed to adults, very little is known about the immune contexture of paediatric malignancies.

Methods: We investigated by gene expression and immunohistochemistry (IHC) the immune microenvironment of five major paediatric cancers: Ewing sarcoma (ES), osteosarcoma (OS), rhabdomyosarcoma (RMS), medulloblastoma (MB) and neuroblastoma (NB; 20 cases each; n = 100 samples total), and correlated them with overall survival.

Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity.

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles.

Characterization of Tumor-immune Microenvironment by High-throughput Image Analysis of CD8 Immunohistochemistry Combined With Modified Masson's Trichrome.

With the advent of checkpoint inhibitors, there is increasing need to study the dynamics of CD8+ T-cells in the tumor microenviroment. In this article, we describe a semi-automated method to quantify and interrogate spatial relationships between T-cells and collagenous stroma in human and mouse tissue samples. The assay combines CD8 immunohistochemistry with modified Masson's trichrome.

Clinical and research applications of multiplexed immunohistochemistry and in situ hybridization.

Over the past decade, invention and adoption of novel multiplexing technologies for tissues have made increasing impacts in basic and translational research and, to a lesser degree, clinical medicine. Platforms capable of highly multiplexed immunohistochemistry or in situ RNA measurements promise evaluation of protein or RNA targets at levels of plex and sensitivity logs above traditional methods - all with preservation of spatial context. These methods promise objective biomarker quantification, markedly increased sensitivity, and single-cell resolution.

Anti-LYPD1/CD3 T-Cell-Dependent Bispecific Antibody for the Treatment of Ovarian Cancer.

Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset.

Embryonic lethality and defective mammary gland development of activator-function impaired conditional knock-in mice.

ERBB3 is a pseudokinase domain-containing member of the ERBB family of receptor tyrosine kinases (RTKs). Following ligand binding, ERBB receptors homo- or hetero-dimerize, leading to a head-to-tail arrangement of the intracellular kinase domains, where the "receiver" kinase domain of one ERBB is activated by the "activator" domain of the other ERBB in the dimer. In ERBB3, a conserved valine at codon 943 (V943) in the kinase C-terminal domain has been shown to be important for its function as an "activator" kinase .

Integrated digital pathology and transcriptome analysis identifies molecular mediators of T-cell exclusion in ovarian cancer.

Close proximity between cytotoxic T lymphocytes and tumour cells is required for effective immunotherapy. However, what controls the spatial distribution of T cells in the tumour microenvironment is not well understood. Here we couple digital pathology and transcriptome analysis on a large ovarian tumour cohort and develop a machine learning approach to molecularly classify and characterize tumour-immune phenotypes.

The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins.

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake.

S100a4 upregulation in Pik3caH1047R;Trp53R270H;MMTV-Cre-driven mammary tumors promotes metastasis.

Background: PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers.

Inhibition of the dipeptidyl peptidase DPP4 (CD26) reveals IL-33-dependent eosinophil-mediated control of tumor growth.

Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors.

New tools for pathology: a user's review of a highly multiplexed method for in situ analysis of protein and RNA expression in tissue.

Tumor cell heterogeneity and tumor cell-stromal interactions are being explored as determinants of disease progression and treatment resistance in solid tumor and hematological malignancies. As such, tools simultaneously capable of highly multiplexed profiling of tissues' protein and RNA content, as well as interrogation of rare or single cells, are required to precisely characterize constituent tumor cell populations, infiltrating lymphocytes and stromal elements. Access to spatial relationships will enable more precise characterization of tumors, support patient stratification and may help to identify novel drug targets.

Actionable Activating Oncogenic ERBB2/HER2 Transmembrane and Juxtamembrane Domain Mutations.

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) that include G660D, R678Q, E693K, and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations.

IFNγ-induced Chemokines Are Required for CXCR3-mediated T-Cell Recruitment and Antitumor Efficacy of Anti-HER2/CD3 Bispecific Antibody.

Purpose: The response to cancer immune therapy is dependent on endogenous tumor-reactive T cells. To bypass this requirement, CD3-bispecific antibodies have been developed to induce a polyclonal T-cell response against the tumor. Anti-HER2/CD3 T-cell-dependent bispecific (TDB) antibody is highly efficacious in the treatment of HER2-overexpressing tumors in mice.

TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells.

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies.

CD8+ T cell infiltration in breast and colon cancer: A histologic and statistical analysis.

The prevalence of cytotoxic tumor infiltrating lymphocytes (TILs) has demonstrated prognostic value in multiple tumor types. In particular, CD8 counts (in combination with CD3 and CD45RO) have been shown to be superior to traditional UICC staging in colon cancer patients and higher total CD8 counts have been associated with better survival in breast cancer patients. However, immune infiltrate heterogeneity can lead to potentially significant misrepresentations of marker prevalence in routine histologic sections.