Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2 þ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8 þ T-cell proliferation and IFNg production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8 þ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

Four 2,3-oxidosqualene analogs, 3, 4, 5, and 6, which are irreversible, time-dependent inhibitors of the enzyme lanosterol synthase, were found to attach covalently within the 231−236 (yeast numbering) segment (Figure ). The attachment was determined by tryptic digestion of the inactivated enzyme, separation of the tryptic cleavage products by C18 reverse phase HPLC, and fragment identification by mass spectroscopy or Edman degradation. W232 and H234 are the targets of the chemical inactivation by cations derived from analogs 3−6. 2,3-Oxidosqualene analogs 7, 8, and 9 inactivated the enzyme with covalent attachment to the 486−512 segment (Figure ), which is in a domain that is predicted to be an amphipathic α-helix. Site-directed mutagenesis of various amino acid residues (76 total) in lanosterol synthase which are conserved in five different species has revealed that residues D456, H146, and H234 are essential for catalytic activity. These and other data permit the formulation of a hypothetical working model of some aspects of the activation and binding of 2,3-oxidosqualene by lanosterol synthase. The model is depicted in Figure . In that model D456 and protonated H146 initiate cyclization, and the domains containing 231−236 and 486−512 make contact with the reacting substrate.

Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme from Saccharomyces cerevisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis, was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps. Using pure enzyme the kinetics of cyclization were determined using Michaelis−Menten analysis for 2,3-oxidosqualene (1) and two analogs in which the C−6 methyl was replaced by H (3) or Cl (4). The measured V max/K M ratios for 1, 3, and 4 were found to be 138, 9.4, and 21.9, respectively, a clear indication that oxirane cleavage and cyclization to form the A-ring are concerted, since the nucleophilicity of the proximate double bond influences the rate of oxirane cleavage. No catalytic metal ions could be detected in purified lanosterol synthase by atomic absorption analysis. Site-directed mutagenesis studies of each of the six strongly conserved aspartic acid residues (D → N mutation) and each of the nine conserved glutamic acid residues (E → Q) revealed that only one, D456, is essential for catalytic function of the enzyme. The essential D456 residue is a likely candidate for electrophilic (specifically protic) activation of the oxirane function.

Indoleamine-2,3-dioxygenase 1 (IDO1) inhibition and its combination with immune checkpoint inhibitors like pembrolizumab have drawn considerable attention from both academia and the pharmaceutical industry. Here, we describe the discovery of a novel class of highly potent IDO1 heme-displacing inhibitors featuring a unique bicyclo[1.1.1]­pentane motif. Compound 1, evolving from an ALIS (automated ligand identification system) hit, exhibited excellent potency but lacked the desired pharmacokinetic profile due to extensive amide hydrolysis of the benzamide moiety. Replacing the central phenyl ring in 1 with a bicyclo[1.1.1]­pentane bioisostere effectively circumvented the amide hydrolysis issue, resulting in the discovery of compound 2 with a favorable overall profile such as excellent potency, selectivity, pharmacokinetics, and a low predicted human dose.

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