Filamin A regulates caspase-3 cleavage in platelets in a protein kinase C (PKC)-dependent manner.

Apoptosis is a critical process for the maintenance of cell populations, and involves mitochondrial depolarization, the sequential cleavage of caspase-9 and -3, followed by the externalization of phosphatidylserine (PS) on the plasma membrane. The actin cytoskeleton and its accessory proteins are known regulators of apoptotic signaling in nucleated cells but their roles in platelet apoptosis are undefined. Filamin A (FLNA) is a ubiquitously expressed actin-crosslinking protein that also serves as an intracellular signaling scaffold. Here we used platelets from mice with a platelet-specific FLNA deficiency (Flnafl/Y, Pf4-cre/+, termed platelet-specific knockout) to test the role of FLNA in platelet apoptosis. Treatment with the BH3-mimetic drug ABT-737 induced caspase-3 cleavage and PS exposure in platelets from floxed mice (Flnafl/Y, termed control) but these effects were essentially abrogated in FLNA-null platelets (platelet-specific knockout). Protein kinase C (PKC), a known FLNA ligand, was also activated by ABT-737, and PKC's phosphorylation of its downstream substrates was attenuated in FLNA-null platelets. The PKC inhibitor bisindolylmaleimide (BIM) also reduced caspase-3 cleavage, thus essentially phenocopying the FLNA-null platelets. Notably, the caspase-3 cleavage defect in FLNA-null platelets was rescued by the PKC-activating phorbol ester PMA, suggesting that FLNA and PKC share a common pathway in regulating platelet apoptosis. Mitochondrial depolarization and caspase-9 cleavage were unaffected by BIM treatment, suggesting that PKC specifically controls the downstream caspase-3 point of the pro-apoptotic signaling pathway. These data point to a novel role for FLNA in the regulation of platelet apoptosis, thus providing an improved understanding of how circulating platelet counts are maintained.

Podocalyxin is required for maintaining blood-brain barrier function during acute inflammation.

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primed PodxlΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

MAPS: machine-assisted phenotype scoring enables rapid functional assessment of genetic variants by high-content microscopy.

BACKGROUND: Genetic testing is widely used in evaluating a patient's predisposition to hereditary diseases. In the case of cancer, when a functionally impactful mutation (i.e. genetic variant) is identified in a disease-relevant gene, the patient is at elevated risk of developing a lesion in their lifetime. Unfortunately, as the rate and coverage of genetic testing has accelerated, our ability to assess the functional status of new variants has fallen behind. Therefore, there is an urgent need for more practical, streamlined and cost-effective methods for classifying variants. RESULTS: To directly address this issue, we designed a new approach that uses alterations in protein subcellular localization as a key indicator of loss of function. Thus, new variants can be rapidly functionalized using high-content microscopy (HCM). To facilitate the analysis of the large amounts of imaging data, we developed a new software toolkit, named MAPS for machine-assisted phenotype scoring, that utilizes deep learning to extract and classify cell-level features. MAPS helps users leverage cloud-based deep learning services that are easy to train and deploy to fit their specific experimental conditions. Model training is code-free and can be done with limited training images. Thus, MAPS allows cell biologists to easily incorporate deep learning into their image analysis pipeline. We demonstrated an effective variant functionalization workflow that integrates HCM and MAPS to assess missense variants of PTEN, a tumor suppressor that is frequently mutated in hereditary and somatic cancers. CONCLUSIONS: This paper presents a new way to rapidly assess variant function using cloud deep learning. Since most tumor suppressors have well-defined subcellular localizations, our approach could be widely applied to functionalize variants of uncertain significance and help improve the utility of genetic testing.

Applied stretch initiates directional invasion through the action of Rap1 GTPase as a tension sensor.

Although it is known that a stiffening of the stroma and the rearrangement of collagen fibers within the extracellular matrix facilitate the movement of tumor cells away from the primary lesion, the underlying mechanisms responsible are not fully understood. We now show that this invasion, which can be initiated by applying tensional loads to a three-dimensional collagen gel matrix in culture, is dependent on the Rap1 GTPases (Rap1a and Rap1b, referred to collectively as Rap1). Under these conditions Rap1 activity stimulates the formation of focal adhesion structures that align with the tensional axis as single tumor cells move into the matrix. These effects are mediated by the ability of Rap1 to induce the polarized polymerization and retrograde flow of actin, which stabilizes integrins and recruits vinculin to preformed adhesions, particularly those near the leading edge of invasive cells. Rap1 activity also contributes to the tension-induced collective invasive elongation of tumor cell clusters and it enhances tumor cell growth in vivo Thus, Rap1 mediates the effects of increased extracellular tension in multiple ways that are capable of contributing to tumor progression when dysregulated.

The cell surface mucin podocalyxin regulates collective breast tumor budding.

BACKGROUND: Overexpression of the transmembrane sialomucin podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if podocalyxin plays a functional role in breast tumor progression. METHODS: MCF-7 breast cancer cells, which express little endogenous podocalyxin, were stably transfected with wild type podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting podocalyxin for stable knockdown. In vitro, the effects of podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice. RESULTS: Forced overexpression of podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of podocalyxin. In three-dimensional (3-D) culture, podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture. CONCLUSIONS: Podocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy.

INTRODUCTION: Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression. METHODS: We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. RESULTS: Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. CONCLUSIONS: We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.

The small molecule genkwanine M induces single mode, mesenchymal tumor cell motility.

Individual tumor cells utilize one of two modes of motility to invade the extracellular matrix, mesenchymal or amoeboid. We have determined that the diterpenoid genkwanine M (GENK) enhances the mesenchymal mode of cell motility that is intrinsic to HT-1080 osteosarcoma cells, stimulates a mesenchymal mode of motility in stationary MDA-MB-453 breast carcinoma cells, and induces a shift to a mesenchymal mode of cell motility in LS174T colorectal adenocarcinoma cells that normally utilize the alternate amoeboid mode of motility. The ability of GENK to stimulate or induce mesenchymal motility was preceded by a rapid cell spreading, elongation and polarization that did not require new gene expression. However, these initial morphologic changes were integrin dependent and they were associated with a reorganization of focal contacts and focal adhesions as well as an activation of the focal adhesion kinase. Therefore, GENK induces a mesenchymal mode of cell motility in a wide variety of tumor cell types that may be mediated, at least in part, by an activation of integrin-associated signaling.

Redistribution of the glycocalyx exposes phagocytic determinants on apoptotic cells.

Phagocytes remove dead and dying cells by engaging "eat-me" ligands such as phosphatidylserine (PtdSer) on the surface of apoptotic targets. However, PtdSer is obscured by the bulky exofacial glycocalyx, which also exposes ligands that activate "don't-eat-me" receptors such as Siglecs. Clearly, unshielding the juxtamembrane "eat-me" ligands is required for the successful engulfment of apoptotic cells, but the mechanisms underlying this process have not been described. Using human and murine cells, we find that apoptosis-induced retraction and weakening of the cytoskeleton that anchors transmembrane proteins cause an inhomogeneous redistribution of the glycocalyx: actin-depleted blebs emerge, lacking the glycocalyx, while the rest of the apoptotic cell body retains sufficient actin to tether the glycocalyx in place. Thus, apoptotic blebs can be engaged by phagocytes and are targeted for engulfment. Therefore, in cells with an elaborate glycocalyx, such as mucinous cancer cells, this "don't-come-close-to-me" barrier must be removed to enable clearance by phagocytosis.

Synthesis and biological studies of neopetrosiamides as inhibitors of cancer cell invasion.

The tricyclic peptides neopetrosiamides A and B, isolated from the marine sponge Neopetrosia sp., are potential antimetastatic agents that inhibit tumour cell invasion by both amoeboid and mesenchymal migration pathways. They differ in the stereochemistry of the methionine sulfoxide at position 24. Our previously reported syntheses using an orthogonal sulfur protection strategy established the critical connectivity of the three disulfide bonds. In this report, fifteen analogues of neopetrosiamide A and B, six which replace selected disulfide bonds and nine which replace the diastereomeric methionine sulfoxide, have been prepared using Fmoc solid-phase peptide chemistry. Disulfide replacement analogues were shown to lose activity, and only one of the methionine sulfoxide analogues retained full bioactivity in morphological studies.

Cofilin-mediated F-actin severing is regulated by the Rap GTPase and controls the cytoskeletal dynamics that drive lymphocyte spreading and BCR microcluster formation.

When lymphocytes encounter APCs bearing cognate Ag, they spread across the surface of the APC to scan for additional Ags. This is followed by membrane contraction and the formation of Ag receptor microclusters that initiate the signaling reactions that lead to lymphocyte activation. Breakdown of the submembrane cytoskeleton is likely to be required for the cytoskeleton reorganization that drives cell spreading and for removing physical barriers that limit Ag receptor mobility. In this report, we show that Ag receptor signaling via the Rap GTPases promotes the dephosphorylation and activation of the actin-severing protein cofilin and that this results in increased severing of cellular actin filaments. Moreover, we show that this cofilin-mediated actin severing is critical for the changes in actin dynamics that drive B and T cell spreading, for the formation of BCR microclusters, and for the increased mobility of BCR microclusters within the plasma membrane after BCR engagement. Finally, using a model APC, we show that activation of this Rap-cofilin signaling module controls the amount of Ag that is gathered into BCR microclusters and that this is directly related to the magnitude of the resulting BCR signaling that is initiated during B cell-APC interactions. Thus, Rap-dependent activation of cofilin is critical for the early cytoskeletal changes and BCR reorganization that are involved in APC-dependent lymphocyte activation.

A tumor-restricted glycoform of podocalyxin is a highly selective marker of immunologically cold high-grade serous ovarian carcinoma.

Introduction: Targeted-immunotherapies such as antibody-drug conjugates (ADC), chimeric antigen receptor (CAR) T cells or bispecific T-cell engagers (eg, BiTE®) all aim to improve cancer treatment by directly targeting cancer cells while sparing healthy tissues. Success of these therapies requires tumor antigens that are abundantly expressed and, ideally, tumor specific. The CD34-related stem cell sialomucin, podocalyxin (PODXL), is a promising target as it is overexpressed on a variety of tumor types and its expression is consistently linked to poor prognosis. However, PODXL is also expressed in healthy tissues including kidney podocytes and endothelia. To circumvent this potential pitfall, we developed an antibody, named PODO447, that selectively targets a tumor-associated glycoform of PODXL. This tumor glycoepitope is expressed by 65% of high-grade serous ovarian carcinoma (HGSOC) tumors. Methods: In this study we characterize these PODO447-expressing tumors as a distinct subset of HGSOC using four different patient cohorts that include pre-chemotherapy, post-neoadjuvant chemotherapy (NACT) and relapsing tumors as well as tumors from various peritoneal locations. Results: We find that the PODO447 epitope expression is similar across tumor locations and negligibly impacted by chemotherapy. Invariably, tumors with high levels of the PODO447 epitope lack infiltrating CD8+ T cells and CD20+ B cells/plasma cells, an immune phenotype consistently associated with poor outcome. Discussion: We conclude that the PODO447 glycoepitope is an excellent biomarker of immune "cold" tumors and a candidate for the development of targeted-therapies for these hard-to-treat cancers.

Expression of the ETV6-NTRK3 gene fusion as a primary event in human secretory breast carcinoma.

We report that human secretory breast carcinoma (SBC), a rare subtype of infiltrating ductal carcinoma, expresses the ETV6-NTRK3 gene fusion previously cloned in pediatric mesenchymal cancers. This gene fusion encodes a chimeric tyrosine kinase with potent transforming activity in fibroblasts. ETV6-NTRK3 expression was confirmed in 12 (92%) of 13 SBC cases, but not in other ductal carcinomas. Retroviral transfer of ETV6-NTRK3 (EN) into murine mammary epithelial cells resulted in transformed cells that readily formed tumors in nude mice. Phenotypically, tumors produced glands and expressed epithelial antigens, confirming that EN transformation is compatible with epithelial differentiation. This represents a recurrent chromosomal rearrangement and expression of a dominantly acting oncogene as a primary event in human breast carcinoma.

Antibody-Drug Conjugates Targeting Tumor-Specific Mucin Glycoepitopes.

Finding the ideal epitope to target is a key element for the development of an antibody-drug conjugate (ADC). To maximize drug delivery to tumor cells and reduce side effects, this epitope should be specific to cancer cells and spare all normal tissue. During cancer progression, glycosylation pathways are frequently altered leading to the generation of new glycosylation patterns selective to cancer cells. Mucins are highly glycosylated proteins frequently expressed on tumors and, thus, ideal presenters of altered glycoepitopes. In this review, we describe three different types of glycoepitopes that are recognized by monoclonal antibodies (mAb) and, therefore, serve as ideal scaffolds for ADC; glycan-only, glycopeptide and shielded-peptide glycoepitopes. We review pre-clinical and clinical results obtained with ADCs targeting glycoepitopes expressed on MUC1 or podocalyxin (Podxl) and two mAbs targeting glycoepitopes expressed on MUC16 or MUC5AC as potential candidates for ADC development. Finally, we discuss current limits in using glycoepitope-targeting ADCs to treat cancer and propose methods to improve their efficacy and specificity.

Modeling Collective Invasion and Single-Cell Mesenchymal Invasion in Three-Dimensional Matrigel-Collagen I Cultures.

Understanding the modes and mechanisms of tumor cell invasion is key to developing targeted therapies against metastatic disease. In vitro assays modeling tumor progression have primarily been optimized for studying classical single-cell migration through an epithelial-mesenchymal transition (EMT). Although experimental and clinical histopathological evidence has revealed that tumor invasion is plastic and that epithelial carcinomas can invade by a range of modes that vary from single, mesenchyme-like cells, all the way to cohesive, collective units, few in vitro assays have been designed to assess these modes specifically. Thus, we have developed a Matrigel-Collagen I overlay assay that is suitable for identifying and quantifying both collective and mesenchymal invasion. This three-dimensional (3D) culture assay utilizes the features of Matrigel and Collagen I to mimic the laminin-rich basement membrane and the stiff, fibrillar Collagen I tumor microenvironment allowing for spheroid invasion to be assessed at the interface between these two matrix components.

Targeting a Tumor-Specific Epitope on Podocalyxin Increases Survival in Human Tumor Preclinical Models.

Podocalyxin (Podxl) is a CD34-related cell surface sialomucin that is normally highly expressed by adult vascular endothelia and kidney podocytes where it plays a key role in blocking adhesion. Importantly, it is also frequently upregulated on a wide array of human tumors and its expression often correlates with poor prognosis. We previously showed that, in xenograft studies, Podxl plays a key role in metastatic disease by making tumor initiating cells more mobile and invasive. Recently, we developed a novel antibody, PODO447, which shows exquisite specificity for a tumor-restricted glycoform of Podxl but does not react with Podxl expressed by normal adult tissue. Here we utilized an array of glycosylation defective cell lines to further define the PODO447 reactive epitope and reveal it as an O-linked core 1 glycan presented in the context of the Podxl peptide backbone. Further, we show that when coupled to monomethyl auristatin E (MMAE) toxic payload, PODO447 functions as a highly specific and effective antibody drug conjugate (ADC) in killing ovarian, pancreatic, glioblastoma and leukemia cell lines in vitro. Finally, we demonstrate PODO447-ADCs are highly effective in targeting human pancreatic and ovarian tumors in xenografted NSG and Nude mouse models. These data reveal PODO447-ADCs as exquisitely tumor-specific and highly efficacious immunotherapeutic reagents for the targeting of human tumors. Thus, PODO447 exhibits the appropriate characteristics for further development as a targeted clinical immunotherapy.

Inflammation-Induced Metastatic Colonization of the Lung Is Facilitated by Hepatocyte Growth Factor-Secreting Monocyte-Derived Macrophages.

A rate-limiting step for circulating tumor cells to colonize distant organ sites is their ability to locate a microenvironmental niche that supports their survival and growth. This can be achieved by features intrinsic to the tumor cells and/or by the conditioning of a "premetastatic" niche. To determine if pulmonary inflammation promotes the latter, we initiated models for inflammatory asthma, hypersensitivity pneumonitis, or bleomycin-induced sterile inflammation before introducing tumor cells with low metastatic potential into the circulation. All types of inflammation increased the end-stage metastatic burden of the lungs 14 days after tumor cell inoculation without overtly affecting tumor extravasation. Instead, the number and size of early micrometastatic lesions found within the interstitial tissues 96 hours after tumor cell inoculation were increased in the inflamed lungs, coincident with increased tumor cell survival and the presence of nearby inflammation-induced monocyte-derived macrophages (MoDM; CD11b+CD11c+). Remarkably, the adoptive transfer of these MoDM was sufficient to increase lung metastasis in the absence of inflammation. These inflammation-induced MoDM secrete a number of growth factors and cytokines, one of which is hepatocyte growth factor (HGF), that augmented tumor cell survival under conditions of stress in vitro. Importantly, blocking HGF signaling with the cMET inhibitor capmatinib abolished inflammation-induced early micrometastatic lesion formation in vivo. These findings indicate that inflammation-induced MoDM and HGF in particular increase the efficiency of early metastatic colonization in the lung by locally preconditioning the microenvironment. IMPLICATIONS: Inflammation preconditions the distant site microenvironment to increase the metastatic potential of tumor cells that arrive there.

Chemical synthesis and biological activity of the neopetrosiamides and their analogues: revision of disulfide bond connectivity.

Neopetrosiamides A and B (2) from the marine sponge Neopetrosia sp. are two diastereomeric tricyclic peptides that inhibit tumor cell invasion associated with metastasis. The reported structures were chemically synthesized using solid-phase peptide synthesis and sequential stepwise disulfide bond formation in solution. The disulfide bond connectivity of the originally proposed structures was revised and confirmed by chemical synthesis together with a combination of HPLC analysis, disulfide mapping, and biological activity testing. This methodology was also utilized to generate analogues containing methionine or norleucine in place of the methionine sulfoxide at position 24. Compounds 4 and 6 demonstrated potent bioactivity comparable to that of the parent peptides.

Primary ovarian mucinous carcinoma of intestinal type: significance of pattern of invasion and immunohistochemical expression profile in a series of 31 cases.

Primary ovarian mucinous carcinomas of the intestinal type are uncommon and earlier reports have included cases diagnosed according to older, less stringent, criteria (which would now be considered borderline tumors) and variable numbers of cases of metastatic adenocarcinoma. This study was conducted to identify all cases of primary mucinous carcinoma of the ovary in a population-based registry, diagnosed according to WHO 2003 criteria, and to characterize their histologic features, immunohistochemical expression profile, and outcome. Thirty-one cases of primary ovarian mucinous carcinoma were included in this study. Immunostaining for 33 markers was performed. Mean age of the patients was 55.4+/-13.5 years. Thirty tumors were stage I or II at presentation. Twenty-six of 31 (83.9%) tumors had expansile stromal invasion, 4 of 31 (12.9%) showed destructive invasion, and 1 of 31 (3.2%) had anaplastic carcinoma in a mural nodule. All cases with destructive invasion showed grade 3 nuclear atypia whereas only 3 of 26 (11.5%) cases with expansile invasion had grade 3 nuclear atypia (P=0.0003). At follow-up, 6 of 26 patients (23.1%) with tumors showing expansile invasion experienced a recurrence, compared with 1 of 4 patients (25%) with destructive invasion and the single patient (100%) with anaplastic carcinoma. There was CK7 positivity in 26 of 31 cases (86.7%), and CK20 and Cdx-2 were each positive in 33.3% of cases. D2-40, calretinin, mesothelin, CA-125, Pax-8, TTF, and WT1 were completely negative in all cases. NHERF1 staining was present in 19 of 26 cases (73%) and its expression was associated with poor prognosis (P=0.05). Our findings support current diagnostic criteria for primary ovarian mucinous carcinoma, that is, the presence of expansile invasion, in the absence of destructive invasion, warrants a diagnosis of carcinoma. A large majority of mucinous carcinomas show only an expansile pattern of invasion and are confined to the pelvis at diagnosis.

A sticky wicket: Defining molecular functions for CD34 in hematopoietic cells.

The CD34 cell surface antigen is widely expressed in tissues on cells with progenitor-like properties and on mature vascular endothelia. In adult human bone marrow, CD34 marks hematopoietic stem and progenitor cells (HSPCs) starting from the bulk of hematopoietic stem cells with long-term repopulating potential (LT-HSCs) throughout expansion and differentiation of oligopotent and unipotent progenitors. CD34 protein surface expression is typically lost as cells mature into terminal effectors. Because of this expression pattern of HSPCs, CD34 has had a central role in the evaluation or selection of donor graft tissue in HSC transplant (HSCT). Given its clinical importance, it is surprising that the biological functions of CD34 are still poorly understood. This enigma is due, in part, to CD34's context-specific role as both a pro-adhesive and anti-adhesive molecule and its potential functional redundancy with other sialomucins. Moreover, there are also critical differences in the regulation of CD34 expression on HSPCs in humans and experimental mice. In this review, we highlight some of the more well-defined functions of CD34 in HSPCs with a focus on proposed functions most relevant to HSCT biology.

PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin.

BACKGROUND: The success of new targeted cancer therapies has been dependent on the identification of tumor-specific antigens. Podocalyxin (Podxl) is upregulated on tumors with high metastatic index and its presence is associated with poor outcome, thus emerging as an important prognostic and theragnostic marker in several human cancers. Moreover, in human tumor xenograft models, Podxl expression promotes tumor growth and metastasis. Although a promising target for immunotherapy, the expression of Podxl on normal vascular endothelia and kidney podocytes could hamper efforts to therapeutically target this molecule. Since pathways regulating post-translational modifications are frequently perturbed in cancer cells, we sought to produce novel anti-Podxl antibodies (Abs) that selectively recognize tumor-restricted glycoepitopes on the extracellular mucin domain of Podxl. METHODS: Splenic B cells were isolated from rabbits immunized with a Podxl-expressing human tumor cell line. Abs from these B cells were screened for potent reactivity to Podxl+ neoplastic cell lines but not Podxl+ primary endothelial cells. Transcripts encoding heavy and light chain variable regions from promising B cells were cloned and expressed as recombinant proteins. Tumor specificity was assessed using primary normal tissue and an ovarian cancer tissue microarray (TMA). Mapping of the tumor-restricted epitope was performed using enzyme-treated human tumor cell lines and a glycan array. RESULTS: One mAb (PODO447) showed strong reactivity with a variety of Podxl+ tumor cell lines but not with normal primary human tissue including Podxl+ kidney podocytes and most vascular endothelia. Screening of an ovarian carcinoma TMA (219 cases) revealed PODO447 reactivity with the majority of tumors, including 65% of the high-grade serous histotype. Subsequent biochemical analyses determined that PODO447 reacts with a highly unusual terminal N-acetylgalactosamine beta-1 (GalNAcß1) motif predominantly found on the Podxl protein core. Finally, Ab-drug conjugates showed specific efficacy in killing tumor cells in vitro. CONCLUSIONS: We have generated a novel and exquisitely tumor-restricted mAb, PODO447, that recognizes a glycoepitope on Podxl expressed at high levels by a variety of tumors including the majority of life-threatening high-grade serous ovarian tumors. Thus, tumor-restricted PODO447 exhibits the appropriate specificity for further development as a targeted immunotherapy.