IMC-Denoise: A content aware denoising pipeline to enhance Imaging Mass Cytometry

Imaging Mass Cytometry (IMC) is an emerging multiplexed imaging technology for analyzing complex microenvironments using more than 40 molecularly-specific channels. However, this modality has unique data processing requirements, particularly for patient tissue specimens where signal-to-noise ratios for markers can be low, despite optimization, and pixel intensity artifacts can deteriorate image quality and downstream analysis. Here we demonstrate an automated content-aware pipeline, IMC-Denoise, to restore IMC images deploying a differential intensity map-based restoration (DIMR) algorithm for removing hot pixels and a self-supervised deep learning algorithm for shot noise image filtering (DeepSNiF). IMC-Denoise outperforms existing methods for adaptive hot pixel and background noise removal, with significant image quality improvement in modeled data and datasets from multiple pathologies. This includes in technically challenging human bone marrow; we achieve noise level reduction of 87% for a 5.6-fold higher contrast-to-noise ratio, and more accurate background noise removal with approximately 2 × improved F1 score. Our approach enhances manual gating and automated phenotyping with cell-scale downstream analyses. Verified by manual annotations, spatial and density analysis for targeted cell groups reveal subtle but significant differences of cell populations in diseased bone marrow. We anticipate that IMC-Denoise will provide similar benefits across mass cytometric applications to more deeply characterize complex tissue microenvironments

Albumin-linked prostate-specific antigen-activated thapsigargin- and niclosamide-based molecular grenades targeting the microenvironment in metastatic castration-resistant prostate cancer

Localized prostate cancer is curable via annihilation of the entire cancer neighborhood by surgery or local radiation. Unfortunately, once metastatic, no available therapy is curative. The vast majority will die despite aggressive systemic combinational androgen-ablation therapies. Thus, there is an urgent need for effective systemic therapeutics that sterilize the entire microenvironment in metastatic castration-resistant prostate cancer (mCRPC). To accomplish this goal, advantage can be taken of the unique biology of mCRPC cells. Like their normal cell of origin, mCRPCs retain expression of the prostate-specific differentiation protein, prostate-specific antigen (PSA), which they abundantly secrete into their extracellular fluid (ECF). This unique, and essentially universal, secretion of enzymatically active PSA into the ECF by mCRPCs creates an exploitable therapeutic index for activation of systemically delivered highly lipophilic toxins as “molecular grenades” covalently linked to cysteine-34 of human serum albumin (HSA) via a stable maleimide containing PSA cleavable peptide such that PSA-dependent hydrolysis (i.e., “detonation”) releases the grenades restrictively within the ECF of mCRPC. This approach decreases dose-limiting host toxicity while enhancing plasma half-life from minutes to days (i.e., pharmacokinetic effect) and increasing the tissue concentration of the maleimide coupled albumin delivery (MAD) in the ECF at sites of cancer due to the enhanced permeability of albumin at these sites (i.e., enhanced permeability and retention effect). This allows the MAD-PSA detonated grenades to circulate throughout the body in a non-toxic form. Only within sites of mCRPC is there a sufficiently high level of enzymatically active PSA to efficiently “pull the pin” on the grenades releasing their lipophilic cell-penetrant toxins from HSA. Thus, if a sufficient level of “detonation” occurs, this will kill mCRPC cells, and sterilize the entire PSA-rich metastatic sites via a bystander effect. In this review, two examples of such MAD-PSA detonated molecular grenades are presented—one based upon thapsigagin and the other on niclosamide. Keywords: Albumin-linked prodrug, Maleimide coupled albumin delivery, Thapsigargin, Niclosamid

Correction: Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for use in Peptide-Prodrug Therapy.

The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 ± 183 M(-1)s(-1)), was further optimized by a P2' N→P substitution yielding L/VP4.1 (kcat/KM = 17446 ± 2203 M(-1)s(-1)). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development

Conceptual schematic of use of an SVV 3C^pro activated peptide prodrug in combination with SVV virotherapy as a novel form of VDEPT.

<p>SVV infects a fraction of tumor cells (1), producing 3C^pro during the viral life cycle. Upon cell lysis, new SVV virions and 3C^pro are released into nearby tissue (2). Administered peptide prodrug would be excluded from cells by the presence of the attached peptide (3), sparing normal tissues, which are non-permissive and therefore cannot express 3C^pro. The 3C^pro present at high concentration exclusively within the tumor microenvironment cleaves this peptide sequence (4), allowing the cytotoxic moiety to enter both infected and adjacent uninfected cells within the tumor, resulting in a powerful local bystander effect (5).</p

Summary of second order rate constants (k_cat/K_M) for the two endogenous substrates, controls and L/VP4 amino acid substitution/truncation mutants.

<p>The altered substrates are shown with the amino acid(s) substitution(s) or truncations shown in bold. Reported values are the average <i>k</i>_cat/<i>K</i>_M values of 5–7 experiments. Uncertainty is expressed by standard deviation.</p><p>Summary of second order rate constants (k_cat/K_M) for the two endogenous substrates, controls and L/VP4 amino acid substitution/truncation mutants.</p

SVV-001 3C^pro substrates are cleaved in the context of a cellular infection.

<p><b>A.</b> Conversion of FRET substrates by SVV-001 3C^pro produced by a cellular SVV infection of permissive SCLC line, NCI-H446. Data points represent the average of three replicates at each time point. The L/VP4.1 FP substrate incubated with uninfected and infected cells, using similar incubations of NHL FP substrate as a negative control. Lines of the same color correspond to curve fits from GraphPad. Error bars on data points were removed for figure clarity. <b>B.</b> Initial reaction rates of L/VP4.1 peptide cleavage by recombinant SVV-001 3C^pro. Data points represent the initial rate of reaction at each concentration of L/VP4.1 peptide calculated from three replicate experiments. Data points were fit to a Michaelis-Menten nonlinear regression from GraphPad and the kinetic constants determined by the curve fit were reported. Standard deviation values of the kinetics constants were calculated by the GraphPad software and propagated through second order rate constant calculations. <b>C.</b> Proteolysis of CPQ2/5-FAM peptides by native SVV-001 3C^pro in a cellular assay with NCI-H446. Data points represent the average relative fluorescence units (RFUs) increase of three replicates at each time point relative to fluorescence at time zero. The L/VP4.1 FQ peptide was incubated with uninfected and infected cells, using similar incubations of NHL FQ peptide as a negative control. Lines of the same color correspond to connecting line between points.</p