Ephrin-B2-expressing natural killer cells induce angiogenesis.

Background: Therapeutic angiogenesis aims to induce new blood vessel growth in ischemic tissues; however, previous clinical trials have had limited success. Studies of uterine angiogenesis revealed a specialized subset of natural killer (NK) cells, called uterine NK (uNK) cells, which have unique proangiogenic abilities. Methods: We show that uNK cells in mice express ephrin-B2, a regulator of angiogenesis, to induce tubule formation in an ex vivo coculture tubule formation assay. We next induced the expression of ephrin-B2 by splenic NK (sNK) cells harvested from male mice. Results: We showed that induced NK (iNK) cells can also instruct endothelial cells to form tubules using ephrin-B2. Conclusions: We concluded that Ephrin-B2 is a marker of proangiogenic uNK cells and that a proangiogenic phenotype characterized by ephrin-B2 can be induced in sNK cells to induce therapeutic angiogenesis.

Clinical molecular genetics evaluation in women with reproductive failures.

Molecular diagnostics is a rapidly growing branch of the clinical laboratory and has accelerated the advance of personalized medicine in the fields of pharmacogenomics, pharmacogenetics, and nutrigenomics. The versatility of molecular biology allows it to be effective in several medical fields that include reproduction, immunogenetics, and virology. Implementation of molecular and sequencing technology in reproductive medicine can add another layer of understanding to better define the causes behind infertility and recurrent reproductive loss. In the following, we examine current molecular methods for probing factors behind reproductive pregnancy loss including reverse transcription polymerase chain reaction and next generation sequencing (NGS). We review several current and potential genetic (DNA) and transcriptional (RNA)-based parameters in women with infertility that can be significant in diagnosis and treatment. These molecular factors can be inferred either from genomic DNA or RNA locally within the endometrium. Furthermore, we consider infection-based abnormalities such as human herpesvirus-6 and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Finally, we present future directions as well as data demonstrating the potential role of human endogenous retroviruses in pregnancy loss. We hope these discussions will assist the clinician in delineating some of the intricate molecular factors that can contribute to infertility and recurrent reproductive failures.

In vivo anti-V-ATPase antibody treatment delays ovarian tumor growth by increasing antitumor immune responses.

Tumor acidity is the key metabolic feature promoting cancer progression and is modulated by pH regulators on a cancer cell's surface that pump out excess protons/lactic acid for cancer cell survival. Neutralizing tumor acidity improves the therapeutic efficacy of current treatments including immunotherapies. Vacuolar-ATPase (V-ATPase) proton pumps encompass unique plasma membrane-associated subunit isoforms, making this molecule an important target for anticancer therapy. Here, we examined the in vivo therapeutic efficacy of an antibody (a2v-mAB) targeting specific V-ATPase-'V0a2' surface isoform in controlling ovarian tumor growth. In vitro a2v-mAb treatment inhibited the proton pump activity in ovarian cancer (OVCA) cells. In vivo intraperitoneal a2v-mAb treatment drastically delayed ovarian tumor growth with no measurable in vivo toxicity in a transplant tumor model. To explore the possible mechanism causing delayed tumor growth, histochemical analysis of the a2v-mAb-treated tumor tissues displayed high immune cell infiltration (M1-macrophages, neutrophils, CD103+ cells, and NK cells) and an enhanced antitumor response (iNOS, IFN-y, IL-1α) compared to control. There was marked decrease in CA-125-positive cancer cells and an enhanced active caspase-3 expression in a2v-mAb-treated tumors. RNA-seq analysis of a2v-mAb tumor tissues further revealed upregulation of apoptosis-related and toll-like receptor pathway-related genes. Indirect coculture of a2v-mAb-treated OVCA cells with human PBMCs in an unbuffered medium led to an enhanced gene expression of antitumor molecules IFN-y, IL-17, and IL-12-A in PBMCs, further validating the in vivo antitumor responses. In conclusion, V-ATPase inhibition using a monoclonal antibody directed against the V0a2 isoform increases antitumor immune responses and could therefore constitute an effective treatment strategy in OVCA.

Iodide Transporters in the Endometrium: A Potential Diagnostic Marker for Women with Recurrent Pregnancy Failures.

OBJECTIVE: The element iodine is an essential nutrient utilized by the thyroid glands, and deficiency of this element has been linked to reproductive failures. Iodide transporters are also present in reproductive tissues and cells of embryonic origin such as the endometrium and trophoblasts, respectively. The aim of this study is to understand if levels of iodide transporters are linked to pregnancy outcomes. SUBJECTS AND METHODS: RNA derived from endometrial biopsies from controls or women with recurrent reproductive failures was analyzed utilizing RT-PCR and targeted RNASeq. RESULTS: When compared to controls, women with 2 or more reproductive failures had a significant increase (>5 fold) in mRNA levels of the iodine transporters NIS and PENDRIN, but not thyroglobulin when probed vis RT-PCR. Targeted RNASeq analysis confirmed these findings when another group of patients were analyzed. CONCLUSION: These findings suggest possible abnormal iodine metabolism and a deficiency of iodine in endometrial tissues from some of the women with reproductive failures. We hypothesize from these findings that inorganic iodide and/or iodine is required for optimal cellular function in reproductive tissues, and that iodide transporters may potentially be used as a marker for infertility or for probing potential localized iodine deficiency that may not present in a typical thyroid panel analysis.

Cancer-associated V-ATPase induces delayed apoptosis of protumorigenic neutrophils.

Tumors and neutrophils undergo an unexpected interaction, in which products released by tumor cells interact to support neutrophils that in turn support cancer growth, angiogenesis, and metastasis. A key protein that is highly expressed by cancer cells in tumors is the a2 isoform V-ATPase (a2V). A peptide from a2V (a2NTD) is secreted specifically by cancer cells, but not normal cells, into the tumor microenvironment. This peptide reprograms neutrophils to promote angiogenesis, cancer cell invasiveness, and neutrophil recruitment. Here, we provide evidence that cancer-associated a2V regulates the life span of protumorigenic neutrophils by influencing the intrinsic pathway of apoptosis. Immunohistochemical analysis of human cancer tissue sections collected from four different organs shows that levels of a2NTD and neutrophil counts are increased in cancer compared with normal tissues. Significant increases in neutrophil counts were present in both poorly and moderately differentiated tumors. In addition, there is a positive correlation between the number of neutrophils and a2NTD expression. Human neutrophils treated with recombinant a2NTD show significantly delayed apoptosis, and such prolonged survival was dependent on NF-κB activation and ROS generation. Induction of antiapoptotic protein expression (Bcl-xL and Bcl-2A1) and decreased expression of proapoptotic proteins (Bax, Apaf-1, caspase-3, caspase-6, and caspase-7) were a hallmark of these treated neutrophils. Autocrine secretion of prosurvival cytokines of TNF-α and IL-8 by treated neutrophils prolongs their survival. Our findings highlight the important role of cancer-associated a2V in regulating protumorigenic innate immunity, identifying a2V as a potential important target for cancer therapy.

Interleukin-22 promotes development of malignant lesions in a mouse model of spontaneous breast cancer.

Interleukin (IL)-22 is recognized as a tumor-supporting cytokine and is implicated in the proliferation of multiple epithelial cancers. In breast cancer, the current knowledge of IL-22 function is based on cell line models and little is known about how IL-22 affects the tumor initiation, proliferation, invasion, and metastasis in the in vivo system. Here, we investigated the tumor stage-specific function of IL-22 in disease development by evaluating the stage-by-stage progression of breast cancer in an IL-22 knockout spontaneous breast cancer mouse model. We found that among all the stages, IL-22 is specifically upregulated in tumor microenvironment (TME) during the malignant transformation stage of breast tumor progression. The deletion of IL-22 gene leads to the arrest of malignant transition stage, and reduced invasion and tumor burden. Administration of recombinant IL-22 in the TME does not influence in vivo tumor initiation and proliferation but only promotes malignant transformation of cancer cells. Mechanistically, deletion of IL-22 gene causes downregulation of epithelial-to-mesenchymal transition (EMT)-associated transcription factors in breast tumors, suggesting EMT as the mechanism of regulation of malignancy by IL-22. Clinically, in human breast tumor tissues, increased number of IL-22+ cells in the TME is associated with an aggressive phenotype of breast cancer. For the first time, this study provides an insight into the tumor stage-specific function of IL-22 in breast tumorigenesis.

Conditional Deletion of the V-ATPase a2-Subunit Disrupts Intrathymic T Cell Development.

Proper orchestration of T lymphocyte development is critical, as T cells underlie nearly all responses of the adaptive immune system. Developing thymocytes differentiate in response to environmental cues carried from cell surface receptors to the nucleus, shaping a distinct transcriptional program that defines their developmental outcome. Our recent work has identified a previously undescribed role for the vacuolar ATPase (V-ATPase) in facilitating the development of murine thymocytes progressing toward the CD4+ and CD8+ αß T cell lineages. Vav1Cre recombinase-mediated deletion of the a2 isoform of the V-ATPase (a2V) in mouse hematopoietic cells leads to a specific and profound loss of peripheral CD4+ and CD8+ αß T cells. Utilizing T cell-restricted LckCre and CD4Cre strains, we further traced this deficiency to the thymus and found that a2V plays a cell-intrinsic role throughout intrathymic development. Loss of a2V manifests as a partial obstruction in the double negative stage of T cell development, and later, a near complete failure of positive selection. These data deepen our understanding of the biological mechanisms that orchestrate T cell development and lend credence to the recent focus on V-ATPase as a potential chemotherapeutic target to combat proliferative potential in T cell lymphoblastic leukemias and autoimmune disease.

Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of Autophagy, Endosome Function, and ERK/MEK Pathway.

Ovarian cancer (OVCA) patients often develop tolerance to standard platinum therapy that accounts for extensive treatment failures. Cisplatin resistant OVCA cells (cis-R) display enhanced survival mechanisms to cope with therapeutic stress. In these cells, increased autophagy process assists in chemoresistance by boosting the nutrient pool under stress. To improve the treatment response, both protective autophagy inhibition and its overactivation are showing efficacy in chemosensitization. Autophagy requires a tightly regulated intracellular pH. Vacuolar ATPases (V-ATPases) are proton extruding nanomotors present on cellular/vesicular membranes where they act as primary pH regulators. V-ATPase 'a2' isoform (V0a2), the major pH sensing unit, is markedly overexpressed on the plasma membrane and the early endosomes of OVCA cells. Previously, V0a2 inhibition sensitized cis-R cells to platinum drugs by acidifying cytosolic pH that elevated DNA damage. Here, we examined how V0a2 inhibition affected endosomal function and the autophagy process as a possible factor for cisplatin sensitization. Clinically, V0a2 expression was significantly higher in tissues from drug nonresponder OVCA patients compared to treatment responders. In vitro V0a2 knockdown in cis-R cells (sh-V0a2-cisR) significantly reduced the tumor sphere-forming ability and caused complete disintegration of the spheres upon cisplatin treatment. The apoptotic capacity of sh-V0a2-cisR improved substantially with potentiation of both intrinsic and extrinsic apoptotic pathway when treated with cisplatin. Unlike the chemical V-ATPase inhibitors that acutely induce autophagy, here, the stable V0a2 inhibition dampened the protective autophagy process in sh-V0a2-cisR cells with downregulated expression of proteins beclin-1, ATG-7, and LC3B and low autophagosome numbers compared to control cis-R cells. These cells showed downregulated ERK/MEK pathway that is known to repress autophagy. Interestingly, upon cisplatin treatment of sh-V0a2-cisR, the autophagy initiation proteins (LC3B, ATG7, and Beclin 1) were found upregulated as a stress response compared to the untreated cells. However, there was a concomitant downstream autophagosome accumulation and an enhanced P62 protein levels indicating the overall block in autophagy flux. Mechanistically, V0a2 knockdown caused defects in early endosome function as the transferrin internalization was impaired. Taken together, this study provides a novel insight into the mechanism by which V-ATPase-isoform regulates autophagy that assists in chemoresistance in ovarian cancer. We conclude that V-ATPase-V0a2 is a potent target for developing an effective treatment to enhance patient survival rates in ovarian cancer.

Hematopoietic stem cell specific V-ATPase controls breast cancer progression and metastasis via cytotoxic T cells.

The interaction of recruited immune effector cells and cancer cells within tumor microenvironment (TME) shapes the fate of cancer progression and metastasis. Many cancers including breast cancer, express a specific vacuolar ATPase (a2V) on their cell surface which acidifies the extracellular milieu helping cancer cell proliferation and metastasis. To understand the role of immune cell-associated-a2V during breast tumor pathogenesis, we knocked-out a2V (KO) from the hematopoietic stem cells (HSC) and generated breast tumors in mice. The a2V-KO mice developed faster growing, larger, and metastatic breast tumors compared to control mice. Further investigation of the TME revealed a significant reduction in the presence of CD4+ and CD8+ T cells in the a2V-KO tumors. Targeted RNA-Seq of the cells of the TME demonstrated that pro-inflammatory cytokines, death receptors, death receptor ligands, and cytotoxic effectors were significantly down-regulated within the a2V-KO TME. Interestingly, analysis of immune cells in the blood, spleen, and thymus of the non-tumor bearing a2V-KO mice revealed a significant decrease in CD4+ and CD8+ T cell populations. For the first time, this study demonstrates that inhibition of V-ATPase expression in HSC leads to a decrease in CD4+ and CD8+ T cell populations and thus promotes breast tumor growth and metastasis.

Dysregulated uterine natural killer cells and vascular remodeling in women with recurrent pregnancy losses.

PROBLEM: Angiogenesis and vascular remodeling in secretory endometrium represent one of the crucial steps in pregnancy establishment, for which uterine NK (uNK) cells have an important role. Impairment of these steps may proceed to implantation and instigate initial pathology of recurrent pregnancy losses (RPL). In this study, we aim to investigate vascular development and density of uNK cells in secretory endometrium of women with RPL. METHODS OF STUDY: Mid-secretory phase endometrial tissues from women with RPL (n = 15) and fertile controls (n = 7) were investigated. CD56+ and CD16+ uNK cells, CD31+ vascular endothelial cells and smooth muscle myosin (SMM)+ . Vascular smooth muscle cells (VSMC) expressing SMM were investigated using immunohistochemistry and western blot. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was used as well. RESULTS: CD56+ uNK number was significantly higher in women with RPL compared to controls (P < 0.0001). uNK cell density by immunohistochemistry was positively correlated with CD56 mRNA expression by qRT-PCR (r2  = 0.43, P = 0.0137). The number of blood vessels represented by the expression of either CD31 or SMM was higher in women with RPL as compared to controls (P < 0.05 and P < 0.0001, respectively), and correlated with the number of uNK cell (r2  = 0.18, P < 0.04, and r2  = 0.65, P < 0.0001, respectively). The wall thickness of spiral arteries was significantly higher in women with RPL as compared with that of controls (P = 0.0027). CONCLUSION: Increased uNK cells in mid-secretory endometrium are associated with increased vascularization and defective vascular transformation of spiral arteries in women with RPL.

Surfactant protein A suppresses preterm delivery induced by live Escherichia coli in mice.

Preterm birth accounts for the majority of neonatal morbidity and mortality in the developed world. A significant proportion of cases of spontaneous preterm labor are attributable to infections within gestational tissues. Surfactant protein A (SP-A), a collectin produced in the fetal lung and other tissues, has been shown previously in mice to suppress preterm delivery due to intrauterine (IU) instillation of sterile proinflammatory substances. Here we report a powerful antilabor effect for SP-A after IU infection with live Escherichia coli. SP-A abolished preterm birth (rate reduced from 100% to 0%) when it was administered into the uterus simultaneously with bacterial infection, reducing it by 75% when administered intravenously at the same time as IU bacterial inoculation, and by 48% when administered intravenously 4 h after IU bacterial infection. This effect on preterm delivery was accompanied by a parallel benefit on fetal survival in utero. SP-A had no effect on bacterial growth but reversed several major consequences of infection, including increased production of inflammatory mediators and a shift in macrophage polarization to the M1 phenotype. These findings suggest that exogenous SP-A has potential use to counteract infection-induced labor by reversing its proinflammatory consequences.

The curious case of vacuolar ATPase: regulation of signaling pathways.

The Vacuolar ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. The structure of V-ATPase has been highly conserved among all eukaryotic cells and is involved in diverse functions across species. V-ATPase is best known for its acidification of endosomes and lysosomes and is also important for luminal acidification of specialized cells. Several reports have suggested the involvement of V-ATPase in maintaining an alkaline intracellular and acidic extracellular pH thereby aiding in proliferation and metastasis of cancer cells respectively. Increased expression of V-ATPase and relocation to the plasma membrane aids in cancer modulates key tumorigenic cell processes like autophagy, Warburg effect, immunomoduation, drug resistance and most importantly cancer cell signaling. In this review, we discuss the direct role of V-ATPase in acidification and indirect regulation of signaling pathways, particularly Notch Signaling.

Interleukin 22 prevents lipopolysaccharide- induced preterm labor in mice.

Preterm birth is widespread and causes 35% of all neonatal deaths. Infants who survive face potential long-term complications. A major contributing factor of preterm birth is infection. We investigated the role of interleukin 22 (IL22) as a potential clinically relevant cytokine during gestational infection. IL22 is an effector molecule secreted by immune cells. While the expression of IL22 was reported in normal nonpregnant endometrium and early pregnancy decidua, little is known about uterine IL22 expression during mid or late gestational stages of pregnancy. Since IL22 has been shown to be an essential mediator in epithelial regeneration and wound repair, we investigated the potential role of IL22 during defense against an inflammatory response at the maternal-fetal interface. We used a well-established model to study infection and infection-associated inflammation during preterm birth in the mouse. We have shown that IL22 is upregulated to respond to an intrauterine lipopolysaccharide administration and plays an important role in controlling the risk of inflammation-induced preterm birth. This paper proposes IL22 as a treatment method to combat infection and prevent preterm birth in susceptible patients.

Mammary epithelium-specific inactivation of V-ATPase reduces stiffness of extracellular matrix and enhances metastasis of breast cancer.

Extracellular matrix (ECM) critically impacts tumor progression and is influenced by both cancer and host tissue cells. While our understanding of cancer cell ECM remodeling is widespread, the importance of host tissue ECM, which provides initial congenial environment for primary tumor formation, is partly understood. Here, we report a novel role of epithelial cell-associated vacuolar ATPase 'a2' isoform (a2V) in regulating breast tissue ECM stiffness to control metastasis. Using a mammary gland-specific a2V-knockout model, we show that in the absence of a2V, breast tumors exhibit atypically soft tumor phenotype, less tumor rigidity, and necrotic tumor microenvironment. These tumors contain a decreased number of cancer cells at primary tumor site, but showed extensive metastases compared to control. Nanomechanical evaluation of normal breast tissues revealed a decrease in stiffness and collagen content in ECM of a2V-deleted breast tissues. Mechanistically, inhibition of a2V expression caused dispersed Golgi morphology with relocation of glycosyltransferase enzymes to early endosomes in mammary epithelial cells. This resulted in defective glycosylation of ECM proteins and production of compromised ECM that further influenced tumor metastasis. Clinically, in patients with cancer, low a2V expression levels in normal breast tissue correlated with lymph node metastasis. Thus, using a new knockout mouse model, we have identified a2V expression in epithelial cells as a key requirement for proper ECM formation in breast tissue and its expression levels can significantly modulate breast tumor dissemination. Evaluation of a2V expression in normal breast tissues can help in identifying patients with high risk of developing metastases.

A Role for Iodide and Thyroglobulin in Modulating the Function of Human Immune Cells.

Iodine is an essential element required for the function of all organ systems. Although the importance of iodine in thyroid hormone synthesis and reproduction is well known, its direct effects on the immune system are elusive. Human leukocytes expressed mRNA of iodide transporters (NIS and PENDRIN) and thyroid-related proteins [thyroglobulin (TG) and thyroid peroxidase (TPO)]. The mRNA levels of PENDRIN and TPO were increased whereas TG transcripts were decreased post leukocyte activation. Flow cytometric analysis revealed that both PENDRIN and NIS were expressed on the surface of leukocyte subsets with the highest expression occurring on monocytes and granulocytes. Treatment of leukocytes with sodium iodide (NaI) resulted in significant changes in immunity-related transcriptome with an emphasis on increased chemokine expression as probed with targeted RNASeq. Similarly, treatment of leukocytes with NaI or Lugol's iodine induced increased protein production of both pro- and anti-inflammatory cytokines. These alterations were not attributed to iodide-induced de novo thyroid hormone synthesis. However, upon incubation with thyroid-derived TG, primary human leukocytes but not Jurkat T cells released thyroxine and triiodothyronine indicating that immune cells could potentially influence thyroid hormone balance. Overall, our studies reveal the novel network between human immune cells and thyroid-related molecules and highlight the importance of iodine in regulating the function of human immune cells.

Microtubule inhibitor, SP-6-27 inhibits angiogenesis and induces apoptosis in ovarian cancer cells.

In ovarian cancer (OVCA), treatment failure due to chemo-resistance is a serious challenge. It is therefore critical to identify new therapies that are effective against resistant tumors and have reduced side effects. We recently identified 4-H-chromenes as tubulin depolymerizing agents that bind to colchicine site of beta-tubulin. Here, we screened a chemical library of substituted 4-H-chromenes and identified SP-6-27 to exhibit most potent anti-proliferative activity towards a panel of human cisplatin sensitive and resistant OVCA cell lines with 50% inhibitory concentration (IC50; mean ± SD) ranging from 0.10 ± 0.01 to 0.84 ± 0.20 µM. SP-6-27 exhibited minimum cytotoxicity to normal ovarian epithelia. A pronounced decrease in microtubule density as well as G2/M cell cycle arrest was observed in SP-6-27 treated cisplatin sensitive/resistant OVCA cells. The molecular mechanism of SP-6-27 induced cell death revealed modulation in cell-cycle regulation by upregulation of growth arrest and DNA damage inducible alpha transcripts (GADD45). An enhanced intrinsic apoptosis was observed in OVCA cells through upregulation of Bax, Apaf-1, caspase-6, -9, and caspase-3. In vitro wound healing assay revealed reduced OVCA cell migration upon SP-6-27 treatment. Additionally, SP-6-27 and cisplatin combinatorial treatment showed enhanced cytotoxicity in chemo-sensitive/resistant OVCA cells. Besides effect on cancer cells, SP-6-27 further restrained angiogenesis by inhibiting capillary tube formation by human umbilical vein endothelial cells (HUVEC). Together, these findings show that the chromene analog SP-6-27 is a novel chemotherapeutic agent that offers important advantages for the treatment of ovarian cancer.

Identification of Novel Bisbenzimidazole Derivatives as Anticancer Vacuolar (H⁺)-ATPase Inhibitors.

The vacuolar (H⁺)-ATPases (V-ATPases) are a family of ATP-driven proton pumps and they have been associated with cancer invasion, metastasis, and drug resistance. Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V) as an initial hit from a similarity search using four known V-ATPase inhibitors (I-IV). Based on the initial hit (V), we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a-e). All newly prepared compounds have been screened for selected human breast cancer (MDA-MB-468, MDA-MB-231, and MCF7) and ovarian cancer (A2780, Cis-A2780, and PA-1) cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e) is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 µM). Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.

Predicting NK cell subsets using gene expression levels in peripheral blood and endometrial biopsy specimens.

PROBLEM: In molecular analysis of tissue biopsy specimens, one crucial aspect is characterization of immune cell populations. This is especially important for evaluation of uterine receptivity by assessing levels of lymphocyte populations including CD56bright CD16- uterine NK cells and CD56dim CD16+ conventional NK cells. Our objective was to investigate whether measuring total RNA transcripts from a tissue specimen would accurately reflect immune cell levels and be a new technique to assess immune cell subsets. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMCs) and endometrial tissues were used. Flow cytometry was utilized for the analysis of lymphocyte subsets in PBMCs, and RT-qPCR was applied to quantify RNA transcripts indicative of lymphocyte and granulocyte populations. RESULTS: In PBMC specimens, there were significant correlations between gene expression levels and cell subsets. NK cells correlated with CD16A, NKp46, and CD56 transcripts, B cells correlated with EBF1, and CD8+ T cells correlated with CD8ß. Finally, endometrial tissues displayed high CD56 expression and very low CD3ε, CD16A, and NKp30, reflecting the characteristic endometrial NK cell subsets. CONCLUSION: Strong correlations between RT-qPCR data and levels of lymphocyte subsets indicate that gene expression analysis will be a useful technique for characterizing levels of CD56+ cells in endometrial tissues.

The immune response in pregnancy and in cancer is active and supportive of placental and tumor cell growth not their destruction.

While many investigators have described the biochemical and physiological similarities between tumor cells and trophoblast cells, in this discourse we will compare primarily their leucocytes, which constitute a large portion of the tumor and its microenvironment as well as the placenta and its microenvironment. There is a remarkable similarity between the cells that support placental growth and development and tumor growth and development. In many cases over half of the cells present in the tumor and the placenta are non-tumor or nontrophoblast cells, immune cells. Most of these immune cells are prevented from attacking the fetal derived placental cells and the self-derived tumor cells. Nevertheless, these leucocytes, in our opinion, are very active and support tumor and placental cell growth through the production of growth factors and angiogenic factors. These cells do this by activating the portion of the immune response which initiates and helps control tissue repair.

Cancer derived peptide of vacuolar ATPase 'a2' isoform promotes neutrophil migration by autocrine secretion of IL-8.

Neutrophils play significant regulatory roles within the tumor microenvironment by directly promoting tumor progression that leads to poor clinical outcomes. Identifying the tumor associated molecules that regulate neutrophil infiltration into tumors may provide new and specific therapeutic targets for cancer treatment. The a2-isoform of vacuolar ATPase (a2V) is uniquely and highly expressed on cancer cell plasma membrane. Cancer cells secrete a peptide from a2V (a2NTD) that promotes the pro-tumorigenic properties of neutrophils. This provides a2V the propensity to control neutrophil migration. Here, we report that the treatment of human neutrophils with recombinant a2NTD leads to neutrophil adherence and polarization. Moreover, a2NTD treatment activates surface adhesion receptors, as well as FAK and Src kinases that are essential regulators of the migration process in neutrophils. Functional analysis reveals that a2NTD can act as a chemo-attractant and promotes neutrophil migration. In addition, a2Neuɸ secrete high levels of IL-8 via NF-κB pathway activation. Confirmatory assays demonstrate that the promoted migration of a2Neuɸ was dependent on the autocrine secretion of IL-8 from a2Neuɸ. These findings demonstrate for the first time the direct regulatory role of cancer associated a2-isoform V-ATPase on neutrophil migration, suggesting a2V as a potential target for cancer therapy.