Mai, Z. | bioRxiv Science

bioRxiv2022

Sparse-spectral microendoscopy for real-time visualization of tumor cell phenotype and microenvironment spatial heterogeneity in vivo

Cancer heterogeneity and its transformation with time propels treatment resistance and confounds patient outcomes. The inability to monitor in vivo the low abundance, heterocellular phenotypes that resist treatment and ultimately lead to patient death limits the ability to design precision therapies. Here we overcome limitations in multiplexed fluorescence phenotyping to introduce real-time, cellular resolution visualization of tumor heterogeneity in vivo. This method was performed to simultaneously map for the first time 5 individual biomarkers of stemness, proliferation, metabolism, leukocytes and angiogenesis deep within the peritoneal cavities of micrometastatic cancer mouse models at 17 frames per second (fps). The newly developed imaging system revealed distinct cancer cell phenotype-immune cell spatial correlations and clearly visualized the dynamic spatial response of resistant cancer cell niches following treatment. Furthermore, wide-field datasets were generated to facilitate derivation of a mathematical framework for quantifying biomarker spatial variation and thereby overcoming the area restrictions of conventional tumor biopsy. These results pave the way for real-time identification of cancer cell phenotypes in a clinical setting, on which optimized treatment regimens can be based for personalized treatment and precision therapy e.g., tumor margin determination during surgical resection. Additionally, this modality can be used to obtain more fundamental insights into tumor heterogeneity and how treatments affect the molecular and cellular responses of patient-specific disease.

bioengineering↗

bioRxiv2022

Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast

With over 3,000 reported structures, monoterpenoid indole alkaloids (MIAs) constitute one of the largest alkaloid groups in nature, including the clinically important anticancer drug vinblastine and its semi-synthetic derivatives from Catharanthus roseus (Madagascars periwinkle). With the elucidation of the complete 28-step biosynthesis for anhydrovinblastine, it is possible to investigate the heterologous production of vinblastine and other medicinal MIAs. In this study, we successfully expressed the flavoenzyme O-acetylstemmadenine oxidase in Saccharomyces cerevisiae (bakers yeast) by signal peptide modification, which is a vinblastine biosynthetic gene that has not been functionally expressed in this system. We also report the simultaneous genomic integration of [~]18 kb MIA biosynthetic gene cassettes as single copies by CRISPR-Cas9 in bakers yeast, which enabled the biosynthesis of vinblastine precursors catharanthine and tabersonine from the feedstocks secologanin and tryptamine. We further demonstrated the biosynthesis of fluorinated and hydroxylated catharanthine and tabersonine derivatives using our yeasts, which showed that the MIA biosynthesis accommodates unnatural substrates, and the system can be further explored to produce other complex MIAs. With over 3,000 members, monoterpenoid indole alkaloids (MIA) are one of the largest and most diverse alkaloids in nature including many human medicines, such as chemotherapeutics vinblastine from Catharanthus roseus (Madagascars periwinkle) and camptothecin from Camptotheca accuminata (happy tree), and antiarrhythmic ajmaline from Rauwolfia serpentina (Indian snakeroot).1 Recent studies have elucidated the complete 28-step biosynthetic pathway for anhydrovinblastine in C. roseus, which involves diverting a primary monoterpene geranyl pyrophosphate into the biosynthesis of secologanin via the iridoid pathway (9 steps), genesis of the first MIA strictosidine that is the universal precursor to almost all MIAs (2 steps), conversion of strictosidine to iboga type MIA catharanthine and aspidosperma type tabersonine (9 steps), decorating tabersonine to vindoline (7 steps), and the final step that couples vindoline and catharanthine to make anhydrovinblastine (Fig. 1). 2-12 These studies not only revealed the remarkable complexity of MIA formations but also enabled the exploration in heterologous production of bioactive MIAs and intermediates that are usually found in low quantities in their natural sources. Notably, strictosidine and a related corynanthe type MIA ajmalicine have been produced de novo in Saccharomyces cerevisiae (bakers yeast), 13,14 while vindoline has been produced in bakers yeast from tabersonine feedstock. 3,15,16 For strictosidine production in yeast, the challenges lie in the generally low monoterpene biosynthesis output and the intermediates consumption by yeast native metabolism.13,14,17 While studies did not report rapid MIA consumption by yeast, vindoline yields were improved by optimizing the stoichiometry of cytochrome P450 monooxygenase (CYP), CYP redox partner CYP reductase (CPR), and other factors related with CYP activities such as endoplasmic reticulum (ER) homeostasis and NADPH co-factor regeneration that are commonly exploited.15,16 In this study, we constructed yeast strains containing the remaining vinblastine biosynthetic segment and produced catharanthine and tabersonine by feeding precursors, secologanin and tryptamine, as well as their unnatural derivatives by feeding substituted tryptamine. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/495323v1_fig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@52eda3org.highwire.dtl.DTLVardef@6f8d6dorg.highwire.dtl.DTLVardef@1ae0e1aorg.highwire.dtl.DTLVardef@1fed9f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO The biosynthetic pathway for monoterpenoid indole alkaloids (MIAs) catharanthine and vindoline in C. roseus, which couple to form the anticancer anhydrovinblastine and other derivatives. TDC: tryptophan decarboxylase (Genbank P17770); STR: strictosidine synthase (Genbank CAA43936); SGD: strictosidine -glucosidase (Genbank AAF28800); GS: geissoschizine synthase (Genbank MF770507); GO: geissoschizine oxidase (Genbank MF770508); Redox1/2: oxidized geissoschizine reductase 1/2 (Genbank MF770509, MF770510); SAT: stemmadenine O-acetyltransferase (Genbank MF770511); ASO: O-acetylstemmadenine oxidase (Genbank MH136588); DPAS: dihydroprecondylocarpine synthase (Genbank A0A1B1FHP3); HL1: hydrolase 1/catharanthine synthase (Genbank MF770512); HL2: hydrolase 2/tabersonine synthase (Genbank MF770513). C_FIG

synthetic biology↗

bioRxiv2021

Landscape of microenvironment in Randall's plaque by single-cell sequencing

Randalls plaque is significantly associated with the occurrence of nephrolithiasis. However, the microenvironment of Randalls plaque is poorly characterized. To investigate the microenvironment of Randalls plaque, we analyzed single-cell RNA data of 3 Randalls plaque and 3 normal renal papillae tissue and identified 11 different cell types. We screened differentially expressed genes among all cell types between Randalls plaque and normal renal papillae. The microenvironment showed two cell types with multiple stone formation-associated transcriptomic programs. Contrary to previous studies, we did not observe macrophage M1/M2 imbalance. Notably, we detected ossification-associated macrophage is enriched in Randalls plaque and validated GPNMB and ACP5 were potential biomarkers on the ossification-associated macrophage. We also identified an endothelial subset harboring active communication (COL15A1+ PCDH17+ endothelial, DPECs) with other cells. Together with Immunofluorescence, we validated ossification-associated macrophage and DPECs are enriched in Randalls plaque tissue. Finally, cell-to-cell communication revealed that Loop of Henle, DPECs, and osteoblasts-associated macrophages was the main source of SPP1 signaling. Our work will further the understanding of the microenvironment among Randalls plaque tissues and provide deep insight into immune modulation.

bioinformatics↗