Highlights d A scRNA-seq study reveals shared and distinct features of human MMRd and MMRp CRC d Co-variation of single-cell transcriptional programs across specimens predicts immune hubs d A myeloid-rich inflammatory hub is identified below the colonic lumen in human CRC d CXCR3-ligand+ cells form foci with activated T cells in human MMRd CRC

Highlights d Cancer-associated fibroblasts contribute to pancreatic cancer heterogeneity d Cancer cells can have a double-positive phenotype: proliferation and invasion d High CAF abundance linked with DP cells enriched for MAPK and STAT3 co-signaling d Intra-tumoral gland types provide tissue heterogeneity linked with clinical outcome

The relationship of SARS-CoV-2 pulmonary infection and severity of disease is not fully understood. Here we show analysis of autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter-patient and intra-patient heterogeneity of pulmonary virus infection. There is a spectrum of high and low virus cases associated with duration of disease. High viral cases have high activation of interferon pathway genes and a predominant M1-like macrophage infiltrate. Low viral cases are more heterogeneous likely reflecting inherent patient differences in the evolution of host response, but there is consistent indication of pulmonary epithelial cell recovery based on napsin A immunohistochemistry and RNA expression of surfactant and mucin genes. Using a digital spatial profiling platform, we find the virus corresponds to distinct spatial expression of interferon response genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.

Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (Ϸ0.03-m 3 ) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the shortwavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.cyanobacteria ͉ photosynthetic pigments ͉ multivariate curve resolution C yanobacteria convert light energy to chemical energy by means of photosynthesis, using water as a source of electrons for CO 2 reduction and O 2 production. A key part of the photosynthesis process is light absorption (harvesting) by pigments, followed by excitation transfer to reaction center chlorophyll (Chl) a of photosystems (PS) II and I (1). These processes take place in thylakoid membranes that in cyanobacteria generally form an extensive internal membrane complex of several layers along the periphery of the cytoplasm, with thylakoids found less frequently toward the center of the cell (2).The pigments associated with the photosynthetic apparatus are bound to thylakoid proteins, modifying their spectral properties and providing a spatial distribution that aids in the efficiency of light harvesting and energy transfer to reaction center Chls. Pigments bound to integral membrane proteins in reaction center complexes in thylakoids of cyanobacteria include Chl a [Ϸ40 per PS II (3) and Ϸ100 per PS I (4)] and carotenoids; the latter act in photoprotection and 3 Chl quenchin...

SignificanceIdentifying predictive biomarkers of therapeutic response for melanoma patients treated with immune checkpoint inhibitors is a major challenge. By combining microfluidic enrichment for melanoma circulating tumor cells (CTCs) together with RNA-based droplet digital PCR quantitation, we have established a highly sensitive and robust platform for noninvasive, blood-based monitoring of tumor burden. Serial monitoring of melanoma patients treated with immune checkpoint inhibitors shows rapid changes in CTC score, which precede standard clinical assessment and are highly predictive of long-term clinical outcome. Early on-treatment digital monitoring of CTC dynamics may thus help identify patients likely to benefit from immune checkpoint inhibition therapy.

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF-mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (TF), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. Significance: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities. This article is highlighted in the In This Issue feature, p. 521

Transcriptional profiling has defined pancreatic ductal adenocarcinoma (PDAC) into distinct subtypes with the majority being classical epithelial (E) or quasi-mesenchymal (QM). Despite clear differences in clinical behavior, growing evidence indicates these subtypes exist on a continuum with features of both subtypes present and suggestive of interconverting cell states. Here, we investigated the impact of different therapies being evaluated in PDAC on the phenotypic spectrum of the E/QM state. We demonstrate using RNA-sequencing and RNA-in situ hybridization (RNA-ISH) that FOLFIRINOX combination chemotherapy induces a common shift of both E and QM PDAC toward a more QM state in cell lines and patient tumors. In contrast, Vitamin D, another drug under clinical investigation in PDAC, induces distinct transcriptional responses in each PDAC subtype, with augmentation of the baseline E and QM state. Importantly, this translates to functional changes that increase metastatic propensity in QM PDAC, but decrease dissemination in E PDAC in vivo models. These data exemplify the importance of both the initial E/QM subtype and the plasticity of E/QM states in PDAC in influencing response to therapy, which highlights their relevance in guiding clinical trials.

The relationship of SARS-CoV-2 lung infection and severity of pulmonary disease is not fully understood. We analyzed autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter- and intra- patient heterogeneity of pulmonary virus infection. There was a spectrum of high and low virus cases that was associated with duration of disease and activation of interferon pathway genes. Using a digital spatial profiling platform, the virus corresponded to distinct spatial expression of interferon response genes and immune checkpoint genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.