whole-cell-electrophysiology-primary-cultured-murine-enterochromaffin

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Mayo Clinic

 Little is known about the role of the immune system in the earliest stages of breast carcinogenesis. We studied quantitative differences in immune cell types between breast tissues from normal donors and those from women with benign breast disease (BBD). A breast tissue matched case-control study was created from donors to the Susan G. Komen for the Cure Tissue Bank (KTB) and from women diagnosed with BBD at Mayo Clinic (Rochester, MN) who either subsequently developed cancer (BBD cases) or remained cancer-free (BBD controls). Serial tissue sections underwent immunostaining and digital quantification of cell number per mm for CD4 T cells, CD8 T cells, CD20 B cells, and CD68 macrophages and quantification of positive pixel measure for CD11c (dendritic cells). In 94 age-matched triplets, BBD lobules showed greater densities of CD8 T cells, CD11c dendritic cells, CD20 B cells, and CD68 macrophages compared with KTB normals. Relative to BBD controls, BBD cases had lower CD20 cell density ( = 0.04). Nearly 42% of BBD cases had no CD20 B cells in evaluated lobules compared with 28% of BBD controls ( = 0.02). The absence of CD20 cells versus the presence in all lobules showed an adjusted OR of 5.7 (95% confidence interval, 1.4-23.1) for subsequent breast cancer risk. Elevated infiltration of both innate and adaptive immune effectors in BBD tissues suggests an immunogenic microenvironment. The reduced B-cell infiltration in women with later breast cancer suggests a role for B cells in preventing disease progression and as a possible biomarker for breast cancer risk. .

Alterations in the Immune Cell Composition in Premalignant Breast Tissue that Precede Breast Cancer Development.

Tumour-targeted immunotherapy offers the unique advantage of specific tumouricidal effects with reduced immune-associated toxicity. However, existing platforms suffer from low potency, inability to generate long-term immune memory and decreased activities against tumour-cell subpopulations with low targeting receptor levels. Here we adopted a modular design approach that uses colloidal nanoparticles as substrates to create a multivalent bi-specific nanobioconjugate engager (mBiNE) to promote selective, immune-mediated eradication of cancer cells. By simultaneously targeting the human epidermal growth factor receptor 2 (HER2) expressed by cancer cells and pro-phagocytosis signalling mediated by calreticulin, the mBiNE stimulated HER2-targeted phagocytosis and produced durable antitumour immune responses against HER2-expressing tumours. Interestingly, although the initial immune activation mediated by the mBiNE was receptor dependent, the subsequent antitumour immunity also generated protective effects against tumour-cell populations that lacked the HER2 receptor. Thus, the mBiNE represents a new targeted, nanomaterial-immunotherapy platform to stimulate innate and adaptive immunity and promote a universal antitumour response.

Multivalent bi-specific nanobioconjugate engager for targeted cancer immunotherapy.

Ligation of programmed cell death-1 (PD-1) in the tumor microenvironment is known to inhibit effective adaptive antitumor immunity. Blockade of PD-1 in humans has resulted in impressive, durable regression responses in select tumor types. However, durable responses have been elusive in ovarian cancer patients. PD-1 was recently shown to be expressed on and thereby impair the functions of tumor-infiltrating murine and human myeloid dendritic cells (TIDC) in ovarian cancer. In the present work, we characterize the regulation of PD-1 expression and the effects of PD-1 blockade on TIDC. Treatment of TIDC and bone marrow-derived dendritic cells (DC) with IL10 led to increased PD-1 expression. Both groups of DCs also responded to PD-1 blockade by increasing production of IL10. Similarly, treatment of ovarian tumor-bearing mice with PD-1 blocking antibody resulted in an increase in IL10 levels in both serum and ascites. While PD-1 blockade or IL10 neutralization as monotherapies were inefficient, combination of these two led to improved survival and delayed tumor growth; this was accompanied by augmented antitumor T- and B-cell responses and decreased infiltration of immunosuppressive MDSC. Taken together, our findings implicate compensatory release of IL10 as one of the adaptive resistance mechanisms that undermine the efficacy of anti-PD-1 (or anti-PD-L1) monotherapies and prompt further studies aimed at identifying such resistance mechanisms. .

IL10 Release upon PD-1 Blockade Sustains Immunosuppression in Ovarian Cancer.

Immune checkpoint inhibition by anti-PDCD1 (anti-PD1) monoclonal antibody has significant therapeutic activity against central nervous system lymphoma in an immunocompetent preclinical model.

While the role of natural killer (NK) cells in breast cancer therapy has been investigated, little information is known about NK cell function and presence in nonmalignant and premalignant breast tissue. Here, we investigate and quantify NK cell marker CD56 and activating ligand MICA in breast tissue with benign breast disease.

CD56+ immune cell infiltration and MICA are decreased in breast lobules with fibrocystic changes.

TAP1-TAP2 heterodimeric complexes are recognized as the transporter associated with antigen processing of major histocompatibility complex class I peptides for recognition by tumor-specific cytotoxic T lymphocytes. In this study, we investigated the immunohistochemical expression of TAP1 and TAP2 in 160 patients with breast cancer and correlated their expression levels with clinicopathologic parameters. The median age of the patient cohort was 52.5 years (range, 30-86 years). Both TAP1 and TAP2 immunohistochemical expression levels correlated significantly with breast cancer characteristics (P < .001). TAP1 expression levels were low to negative in stage I breast tumors. TAP1 and TAP2 levels were significantly higher in grade 3 tumors than low-grade (grade 1 and 2) tumors. TAP1 and TAP2 expression levels were not significantly different among different levels of HER2-expressing tumors and did not vary by estrogen and progesterone receptor status or patient age. Both TAP1 and TAP2 overexpression in breast cancer might be an indicator of an aggressive breast tumor.

Downregulation of TAP1 and TAP2 in early stage breast cancer.

In the gastrointestinal (GI) epithelium, enterochromaffin (EC) cells are enteroendocrine cells responsible for producing >90% of the body's serotonin (5-hydroxytryptamine, 5-HT). However, the molecular mechanisms of EC cell function are poorly understood. Here, we found that EC cells in mouse primary cultures fired spontaneous bursts of action potentials. We examined the repertoire of voltage-gated sodium channels (Na) in fluorescence-sorted mouse EC cells and found that Scn3a was highly expressed. Scn3a-encoded Na1.3 was specifically and densely expressed at the basal side of both human and mouse EC cells. Using electrophysiology, we found that EC cells expressed robust Na1.3 currents, as determined by their biophysical and pharmacologic properties. Na1.3 was not only critical for generating action potentials in EC cells, but it was also important for regulating 5-HT release by these cells. Therefore, EC cells use Scn3a-encoded voltage-gated sodium channel Na1.3 for electrical excitability and 5-HT release. Na1.3-dependent electrical excitability and its contribution to 5-HT release is a novel mechanism of EC cell function.

Sodium channel Na1.3 is important for enterochromaffin cell excitability and serotonin release.

In breast adipose tissue, macrophages that encircle damaged adipocytes form "crown-like structures of breast" (CLS-B). Although CLS-B have been associated with breast cancer, their role in benign breast disease (BBD) and early carcinogenesis is not understood. We evaluated breast biopsies from three age-matched groups ( = 86 each, mean age 55 years), including normal tissue donors of the Susan G. Komen for the Cure Tissue Bank (KTB), and subjects in the Mayo Clinic Benign Breast Disease Cohort who developed cancer (BBD cases) or did not develop cancer (BBD controls, median follow-up 14 years). Biopsies were classified into histologic categories, and CD68-immunostained tissue sections were evaluated for the frequency and density of CLS-B. Our data demonstrate that CLS-B are associated with BBD: CLS-B-positive samples were significantly less frequent among KTB biopsies (3/86, 3.5%) than BBD controls (16/86 = 18.6%,  = 0.01) and BBD cases (21/86 = 24%,  = 0.002). CLS-B were strongly associated with body mass index (BMI); BMI < 25: 7% CLS-B positive, BMI 25-29: 13%, and BMI ≥ 30: 29% ( = 0.0005). Among BBD biopsies, a high CLS-B count [>5 CLS-B/sample: 10.5% (BBD cases) vs 4.7% (BBD controls),  = 0.007] conferred a breast cancer OR of 6.8 (95% CI, 1.4-32.4),  = 0.02, after adjusting for adipose tissue area (cm), histologic impression, and BMI. As high CLS-B densities are independently associated with an increased breast cancer risk, they may be a promising histologic marker of breast cancer risk in BBD. .

Macrophagic "Crown-like Structures" Are Associated with an Increased Risk of Breast Cancer in Benign Breast Disease.

Malignant cells have the capacity to rapidly grow exponentially and spread in part by suppressing, evading, and exploiting the host immune system. Immunotherapy is a form of oncologic treatment directed towards enhancing the host immune system against cancer. In recent years, manipulation of immune checkpoints or pathways has emerged as an important and effective form of immunotherapy. Agents that target cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1 (PD-L1) are the most widely studied and recognized. Immunotherapy, however, extends beyond immune checkpoint therapy by using new molecules such as chimeric monoclonal antibodies and antibody drug conjugates that target malignant cells and promote their destruction. Genetically modified T cells expressing chimeric antigen receptors are able to recognize specific antigens on cancer cells and subsequently activate the immune system. Native or genetically modified viruses with oncolytic activity are of great interest as, besides destroying malignant cells, they can increase anti-tumor activity in response to the release of new antigens and danger signals as a result of infection and tumor cell lysis. Vaccines are also being explored, either in the form of autologous or allogenic tumor peptide antigens, genetically modified dendritic cells that express tumor peptides, or even in the use of RNA, DNA, bacteria, or virus as vectors of specific tumor markers. Most of these agents are yet under development, but they promise to be important options to boost the host immune system to control and eliminate malignancy. In this review, we have provided detailed discussion of different forms of immunotherapy agents other than checkpoint-modifying drugs. The specific focus of this manuscript is to include first-in-human phase I and phase I/II clinical trials intended to allow the identification of those drugs that most likely will continue to develop and possibly join the immunotherapeutic arsenal in a near future.

Cancer immunotherapy beyond immune checkpoint inhibitors.

Immune checkpoints consist of inhibitory and stimulatory pathways that maintain self-tolerance and assist with immune response. In cancer, immune checkpoint pathways are often activated to inhibit the nascent anti-tumor immune response. Immune checkpoint therapies act by blocking or stimulating these pathways and enhance the body's immunological activity against tumors. Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1(PD-L1) are the most widely studied and recognized inhibitory checkpoint pathways. Drugs blocking these pathways are currently utilized for a wide variety of malignancies and have demonstrated durable clinical activities in a subset of cancer patients. This approach is rapidly extending beyond CTLA-4 and PD-1/PD-L1. New inhibitory pathways are under investigation, and drugs blocking LAG-3, TIM-3, TIGIT, VISTA, or B7/H3 are being investigated. Furthermore, agonists of stimulatory checkpoint pathways such as OX40, ICOS, GITR, 4-1BB, CD40, or molecules targeting tumor microenvironment components like IDO or TLR are under investigation. In this article, we have provided a comprehensive review of immune checkpoint pathways involved in cancer immunotherapy, and discuss their mechanisms and the therapeutic interventions currently under investigation in phase I/II clinical trials. We also reviewed the limitations, toxicities, and challenges and outline the possible future research directions.

Next generation of immune checkpoint therapy in cancer: new developments and challenges.

 Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients. We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later. Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months. The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. .

Folate Receptor Alpha Peptide Vaccine Generates Immunity in Breast and Ovarian Cancer Patients.

Recurrent  fusions in indolent T-cell lymphoproliferative disorder of the gastrointestinal tract.

Resected HER2 breast cancer patients treated with adjuvant trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. We previously showed that trastuzumab and chemotherapy induce HER2-specific antibodies which correlate with improved survival in HER2 metastatic breast cancer patients. It remains unclear whether the generation of immunity required trastuzumab and whether endogenous antibody immunity is associated with improved disease-free survival in the adjuvant setting. In this study, we addressed this question by analyzing serum anti-HER2 antibodies from a subset of patients enrolled in the NCCTG trial N9831, which includes an arm (Arm A) in which trastuzumab was not used. Arms B and C received trastuzumab sequentially or concurrently to chemotherapy, respectively.

Generation of HER2-specific antibody immunity during trastuzumab adjuvant therapy associates with reduced relapse in resected HER2 breast cancer.

Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces, and thereby regulate intestinal fluid secretion. However, it is unknown whether EE and EC cells are directly mechanosensitive, and if so, what the molecular mechanism of their mechanosensitivity is. Consequently, the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors, and they are expressed in mouse and human EC cells. Here, we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells, and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current, which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca increase in isolated cells as well as in EE cells within intestinal organoids, and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive, uncovers Piezo2 as their primary mechanotransducer, defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release, and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology.

Katilyn Knutson

Enteric Neuroscience Program,
Division of Gastroenterology & Hepatology,
Department of Physiology & Biomedical Engineering,
Enteric Neuroscience Program, Division of Gastroenterology & Hepatology,Department of Physiology & Biomedical Engineering

Mayo Clinic

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells