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The ICE3 trial is designed to evaluate the safety and efficacy of breast cryoablation, enabling women older than 60 years with low-risk early-stage breast cancers to benefit from a nonsurgical treatment and to avoid the associated surgical risks. The ICE3 trial is a prospective, multi-center, single-arm, non-randomized trial including women age 60 years or older with unifocal, ultrasound-visible invasive ductal carcinoma size 1.5 cm or smaller and classified as low to intermediate grade, hormone receptor (HR)-positive, and human epidermal growth factor receptor 2 (HER2)-negative. Ipsilateral breast tumor recurrence (IBTR) at 5 years was the primary outcome. A 3-year interim analysis of IBTR was performed, and the IBTR probability was estimated using the Kaplan-Meier method. Full eligibility for the study was met by 194 patients, who received successful cryoablation per protocol. The mean age was 75 years (range, 55–94 years). The mean tumor length was 8.1 mm (range, 8–14.9 mm), and the mean tumor width was 7.4 mm (range, 2.8–14 mm). During a mean follow-up period of 34.83 months, the IBTR rate was 2.06% (4/194 patients). Device-related adverse events were reported as mild in 18.4% and moderate in 2.4% of the patients. No severe device-related adverse events were reported. More than 95% of the patients and 98% of the physicians reported satisfaction with the cosmetic results at the clinical follow-up evaluation. Breast cryoablation presents a promising alternative to surgery while offering the benefits of a minimally invasive procedure with minimal risks. Further study within a clinical trial or registry is needed to confirm cryoablation as a viable alternative to surgical excision for appropriately selected low-risk patients.

Background Atrioventricular valve development relies upon the precisely defined dimensions of the atrioventricular canal (AVC). Current models suggest that Wnt signaling plays an important role atop a pathway that promotes AVC development. The factors that confine AVC differentiation to the appropriate location, however, are less well understood. Results Transmembrane protein 2 (Tmem2) is a key player in restricting AVC differentiation: in zebrafish, tmem2 mutants display an expansion of AVC characteristics, but the molecular mechanism of Tmem2 function in this context remains unclear. Through structure-function analysis, we demonstrate that the extracellular portion of Tmem2 is crucial for its role in restricting AVC boundaries. Importantly, the Tmem2 ectodomain contains regions implicated in the depolymerization of hyaluronic acid (HA). We find that tmem2 mutant hearts exhibit excess HA deposition alongside broadened distribution of Wnt signaling. Moreover, addition of ectopic hyaluronidase can restore the restriction of AVC differentiation in tmem2 mutants. Finally, we show that alteration of a residue important for HA depolymerization impairs the efficacy of Tmem2 function during AVC development. Conclusions Taken together, our data support a model in which HA degradation, regulated by Tmem2, limits the distribution of Wnt signaling and thereby confines the differentiation of the AVC.

Background Spatially resolved gene expression has emerged as a crucial technique to understand complex multicellular interactions within the tumor and its microenvironment. Interrogation of complex cellular interactions within the tumor microenvironment (TME) requires a multi-omics approach where multiple RNA and protein targets can be visualized within the same tumor sample and be feasible in FFPE sample types. Simultaneous detection of RNA and protein can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. Examination of RNA by in situ hybridization (ISH) and protein by immunohistochemistry (IHC) or immunofluorescence (IF) are widely used and accepted techniques for the detection of biomarkers in tumor samples. Given the similarities in workflow, co-detection of RNA and protein by combining ISH and IHC/IF in a single assay can be a powerful multi-omics solution for interrogating the complex tumor and its microenvironment. Methods In this report we combined the single cell, single molecule RNA ISH technology known as RNAscope with IHC/IF to simultaneously detect RNA and protein in the same FFPE tumor section using both chromogenic and fluorescence detection methods. Results We demonstrate co-localization of target mRNA and the corresponding protein in human cancer samples, visualize infiltration of immune cells into the TME, characterize the activation state of immune cells in the TME, identify single cell gene expression within cellular boundaries demarcated by IHC/IF, examine cell type-specific expression of multiple immune checkpoint markers, and distinguish endogenous T cells from activated CAR+ T cells. Overall, we show that co-detection of RNA by the RNAscope ISH assay and protein by the IHC/IF assay in the same FFPE section is a feasible methodology. The combined RNAscope ISH-IHC/IF workflow is a powerful technique that can be used to study gene expression signatures at the RNA and protein level with spatial and single cell resolution. Conclusions By leveraging the strength of the similar workflows of RNAscope ISH and IHC/IF assays, this methodology combines transcriptomics and proteomics in the same tissue section, providing a multi-omics approach for characterizing complex tissues and revealing cell type specific gene expression with spatial and single cell resolution.

Complex tissues such as tumors are comprised of multiple cells types and extracellular matrix. These cells include heterogenous populations of immune cells that infiltrate the tumors. Understanding the composition of these immune infiltrates in the tumor microenvironment (TME) can provide key insights to guide therapeutic intervention and predict treatment response. Thorough understanding of complex tissue dynamics and immune cell characterization requires a multi-omics approach. Simultaneous detection of RNA and protein using in situ hybridization (ISH) and immunohistochemistry/immunofluorescence (IHC/IF) can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. However, a sequential workflow of ISH followed by IHC/IF frequently yields suboptimal protein detection because the protease digestion step in the ISH protocol resulting in poor antibody signal. Here we demonstrate a newly developed integrated ISH/IHC workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell resolution with spatial and morphological context. To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using specific antibodies against CD68, CD8, CD4 and CD56, respectively. Precise characterization of these immune cells was achieved by using probes against targets such as CCL5, IFNG, GNZB, IL-12, NCR1 etc. that not only help in identifying specific immune cells but also assist in determining their activation states. We identified subsets of T cells such as CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we were able to determine the activation states of CD8+ T cells by visualizing the expression of IFNG and GZMB. Furthermore, infiltrating macrophages were identified by detecting the CD68 protein expression while the M1 and M2 subsets were differentiated by detecting the M2-specific target RNA for CD163. Similarly, NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. Interestingly, the degree of infiltration of the different immune cell populations varied based on the tumor type. In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies - including many previously incompatible antibodies - with RNAscope. This new workflow provides a powerful new approach to identifying and characterizing tumor infiltrating populations of immune cells. Citation Format: Anushka Dikshit, Jyoti Phatak, Lydia Hernandez, Emerald Doolittle, Vasudha Murlidhar, Bingqing Zhang, Xiao-Jun Ma. Characterizing tumor-infiltrated immune cells with spatial context using an integrated RNAscope-immunohistochemistry co-detection workflow in FFPE tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB235.

Characterizing heterogenous populations of tumor-infiltrating immune cells requires a multi-omics approach. Here we demonstrate a newly developed integrated in situ hybridization (ISH) and immunohistochemistry (IHC/IF) workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell and spatial resolution. To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using antibodies against CD68, CD8, CD4 and CD56 in combination with probes targeting CCL5, IFNG, GNZB, IL-12, NCR1 etc. We identified CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we determine the activation states of CD8+ T cells by visualizing IFNG and GZMB expression. Furthermore, infiltrating macrophages were detected by CD68 protein expression while the M1 and M2 subsets were differentiated by using the M2-specific marker, CD163. NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. The degree of immune cell infiltration varied based on the tumor type. In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies, including many previously incompatible antibodies with RNAscope . This new workflow provides a powerful approach to identifying and characterizing tumor infiltrating immune cells.

Spatially resolved gene expression has emerged as a crucial technique to understand complex multicellular interactions within the tumor and its microenvironment. Interrogation of complex cellular interactions within the tumor microenvironment (TME) requires a multi-omics approach where multiple RNA and protein targets can be visualized within the same tumor sample and be feasible in FFPE sample types. Simultaneous detection of RNA and protein can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. Examination of RNA by in situ hybridization (ISH) and protein by immunohistochemistry (IHC) or immunofluorescence (IF) are widely used and accepted techniques for the detection of biomarkers in tumor samples. Given the similarities in workflow, co-detection of RNA and protein by combining ISH and IHC/IF in a single assay can be a powerful multi-omics solution for interrogating the complex tumor and its microenvironment. In this report we combined the single cell, single molecule RNA ISH technology known as RNAscope with IHC/IF to simultaneously detect RNA and protein in the same FFPE tumor section using both chromogenic and fluorescence detection methods. We demonstrate co-localization of target mRNA and the corresponding protein in human cancer samples, visualize infiltration of immune cells into the TME, characterize the activation state of immune cells in the TME, identify single cell gene expression within cellular boundaries demarcated by IHC/IF, examine cell type-specific expression of multiple immune checkpoint markers, and distinguish endogenous T cells from activated CAR+ T cells. Overall, we show that co-detection of RNA by the RNAscope ISH assay and protein by the IHC/IF assay in the same FFPE section is a feasible methodology. The combined RNAscope ISH-IHC/IF workflow is a powerful technique that can be used to study gene expression signatures at the RNA and protein level with spatial and single cell resolution. By leveraging the strength of the similar workflows of RNAscope ISH and IHC/IF assays, this methodology combines transcriptomics and proteomics in the same tissue section, providing a multi-omics approach for characterizing complex tissues and revealing cell type specific gene expression with spatial and single cell resolution. Citation Format: Anushka Dikshit, Jyoti Phatak, Siobhan Kernag, Helly Pimental, Hailing Zong, Courtney Todorov, Lydia Hernandez, Jeffrey Kim, Bingqing Zhang, Courtney Anderson, Xiao-Jun Ma. Spatially resolve RNA and protein simultaneously in FFPE tumor samples by combining RNAscope in situ hybridization and immunohistochemistry assays [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2706.

Head and neck squamous cell carcinoma (HNSCC) is the most prevalent cancer affecting the upper aerodigestive tract, with an annual incidence of 600,000 patients and a five year survival of approximately 60% worldwide. Molecular mechanisms driving the development of HNSCC have recently begun to be discovered, with The Cancer Genome Atlas (TCGA) uncovering the genomic landscape of 279 cases of HNSCC. Alterations in cell death pathways were commonly found in the TCGA analysis, with ∼30% of samples harboring 11q13/22 amplifications and overexpression of genes encoding for Fas-associated death domain (FADD) and/or cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2). While overexpression of cIAP1 has been implicated in resistance to cytotoxic therapies, the role of FADD amplification as a target for therapy and in mechanisms of cell death is not well understood. Birinapant is a novel second mitochondria-derived activator of caspases (SMAC)-mimetic that targets and promotes degradation of cIAPs. Its clinical efficacy is currently being investigated in phase II trials of patients with ovarian cancer and leukemia. However, its preclinical and clinical efficacies have not been tested in HNSCC and genomic markers of sensitivity remain to be defined. Here we hypothesized that overexpression of FADD and cIAP1/2 could modulate birinapant sensitivity in HNSCC. To test this hypothesis, we have treated a panel of 11 HPV(-) and 8 HPV(+) HNSCC cell lines with birinapant alone and in combination with death agonists TNFα or TRAIL. UMSCC-46, an HPV(-) cell line which possesses high FADD expression, was the only cell line to reach half maximal inhibitory concentration (IC50) 72 hours post treatment with birinapant alone (IC50 = 10.7 nM); however, 8 of 11 HPV(-) cell lines and all 8 HPV(+) cell lines attained an IC50 (range: 0.1 - 794 nM) when treated with birinapant in combination with either TNFα or TRAIL. We further demonstrated that forced FADD overexpression in a previously resistant cell line (UMSCC-38) led to sensitization when treated with birinapant and TNFα. In vivo, two FADD/cIAP1 overexpressing murine xenograft models of HNSCC, UMSCC-46 and UMSCC-11B, were treated with birinapant at 15 mg/kg or 30 mg/kg every 3 days for a total of 10 treatments. The single modality regimen led to tumor growth inhibition and prolonged host survival. Additionally, combination treatment with birinapant 15 mg/kg and radiation 2Gy/day M-F for 2 weeks synergistically induced TNFα and led to a cure of animals bearing UMSCC-46 xenografts. Mechanistically, birinapant enhanced degradation of cIAP1 and modulated caspase apoptotic or MLKL necroptotic cell death markers in vitro and in vivo. These results suggest that patients harboring genomic alterations in FADD and/or cIAP overexpression may be candidates for treatment with birinapant and radiation. Supported by NIDCD intramural projects ZIA-DC-000073, and 74. Citation Format: Adeeb Derakhshan, Danielle Eytan, Grace Snow, Sophie Carlson, Anthony Saleh, Hui Cheng, Stephen Schiltz, Suresh Mohan, Shaleeka Cornelius, Jamie Coupar, Anastasia Sowers, Lydia Hernandez, James Mitchell, Christina Annunziata, Zhong Chen, Carter Van Waes. Targeted therapy for head and neck squamous cell carcinoma using the novel SMAC-mimetic birinapant. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3821.

Electrocoagulation (EC) is a wastewater treatment process in which aqueous pollutants can be removed by adsorption, entrapment, precipitation or coalescence during a coagulation step produced by electrochemically generated metallic species. When using Fe as the sacrificial electrode, Fe2+ and Fe3+ ions are formed. As Fe3+ species are paramagnetic, this property can in principle be used to facilitate their removal through the application of a magnetic field. In the present work we present a proof-of-concept for a combined electrochemical-magnetic method for pollutant removal. For this approach, the amounts of Fe2+ and Fe3+ produced in an EC cell at various voltages were measured by spectroscopic methods to confirm that Fe3+ species predominate (up to 84%). The effectiveness of the presence of a magnetic field in the precipitation of coagulants from a suspension was confirmed by monitoring the turbidity change versus time with and without exposure to a magnetic field, up to a 30% improvement.

We have demonstrated the selective secretion of high mol wt PRL series (big big PRL) in women with hyperprolactinemia and normal ovarian function. This observation suggests that big big PRL is immunologically similar, but biologically less active, than monomeric or little PRL. In this study we determined the molecular size heterogeneity of immunoreactive PRL in the serum from two ovulatory hyperprolactinemic women (subjects A and B) who had large amounts of serum big big PRL during a menstrual cycle and/or gestation. Serum samples obtained throughout the menstrual cycle (days 6, 10, 14, 17, 23, and 28, taking as day 1 the first day of bleeding) and pregnancy (weeks 7, 9, 11, 15, 20, 25, 30, 34, and 38) were fractionated by gel filtration chromatography. PRL was identified in column eluates by specific RIA. Two additional pregnant women, one with a bromocriptine-treated PRL-secreting adenoma (subject C), and a normal woman (subject D) were studied. Big big PRL was the predominant species throughout the different phases of the menstrual cycle in subject B, comprising 70-80% of the total immunoreactive PRL. Most of the remainder was big PRL, and little PRL was present in only small amounts (6-12%) during the luteal phase. During their pregnancies, the serum PRL in subjects A and B initially was mostly big big PRL, but later in gestation the PRL composition shifted from the high mol wt variants to little PRL. The infant's cord (subject A) and peripheral (subject B) serum at birth contained appreciable quantities of big big and big PRL, respectively. These results indicate that structural changes in PRL occur during pregnancy and the menstrual cycle which are probably influenced by the hormonal environment. In addition, the occurrence of larger mol wt PRL species in the serum of the infant of a hyperprolactinemic mother suggests that the presence of high proportions of big big PRL in the serum is genetically determined.

The subfornical organ (SFO), one of the brain circumventricular organs, contains immuno-reactive arginine8-vasopressin (AVP). AVP in the SFO area may originate in neurons of the hypothalamo-neurohypophysial system. If AVP in the SFO area is part of the magnocellular neuroendocrine system and is important in the regulation of hydration, then its concentration ([]) should change during prolonged dehydration. The SFO is also a target for angiotensin II when the peptide stimulates drinking and releases AVP from the hypothalamo-neurohypophysial system. For these reasons, it was reasoned that hormones of the renin-angiotensin system may also influence [AVP] in the SFO area. To test these hypotheses [AVP] was measured in the SFO area, hippocampal commissure-fornix (HC-F), neural lobe and plasma of rats after 24, 48 and 72 h of water deprivation and at various times after intracerebroventricular (i.v.t.) administration of 5 milli-Goldblatt Units of renin. Before examining the responsiveness of [AVP] in these brain regions to stimulation, we characterized the extraction and recovery of AVP from brain tissue and determined the variance of [AVP] in the SFO area and HC-F among different groups of animals. AVP was extracted from pooled brain tissue into 0.1 N HCl. The homogenate was centrifuged and AVP in the supernatant was quantified by radioimmunoassay either directly or after bentonite extraction. AVP extracted from the SFO area and HC-F displaced labelled antigen bound to antisera in a manner similar to that displaced by standard AVP. The recovery of AVP, added to the 0.1 N HCl extract and assayed directly, averaged 78–108%, whereas 51% was recovered after bentonite extraction. [AVP] in the SFO area from 47 or more groups of 2–6 organs each, averaged 16 ± 5pg/organ, 16 ± 4pg/mg wet wt, 153 ± 31pg/mg protein, and was not significantly different from that contained in the HC-F, 10 ± 1pg/mg wet wt and 111 ± 16pg/mg protein. A frequency histogram of these data revealed a normal (HC-F) and skewed distribution (SFO). Water deprivation for 24, 48 and 72 h stimulated drinking and increased plasma [AVP]. The elevation in plasma [AVP] plateaued after 48 and 72 h of water deprivation, whereas [AVP] in the neural lobe was reduced (P < 0.05). Water deprivation increased [AVP] in the HC-F (control vs 72 h water deprivation), but did not alter hormone in the SFO area when expressed as pg/mg protein or pg/mg wet wt. Renin elevated [AVP] in plasma for 3 h and stimulated drinking behavior for 6 h after i.v.t. administration. [AVP] in the SFO area, HC-F or neural lobe did not change 5, 30, 180 or 360 min after i.v.t. renin. The observation that the concentration of AVP in the SFO remains relatively constant after treatments which markedly alter hydration supports the conclusion that the peptide in the SFO subserved some other function. Alternately, a dynamic balance between synthesis and degradation of AVP, consistent with rapid turnover during changes in hydration, is possible.