Cell lineage tracing links ERα loss in Erbb2-positive breast cancers to the arising of a highly aggressive breast cancer subtype.

HER2-positive (HER2+) breast cancers (BrCs) contain approximately equal numbers of ERα+HER2+ and ERα-HER2+ cases. An enduring obstacle is the unclear cell lineage-related characteristics of these BrCs. Although ERα+HER2+ BrCs could lose ERα to become ERα-HER2+ BrCs, direct evidence is missing. To investigate ERα dependencies and their implications during BrC growth and metastasis, we generated ERαCreRFP-T mice that produce an RFP-marked ERα+ mammary gland epithelial cell (MGEC) lineage. RCAS virus-mediated expression of Erbb2, a rodent Her2 homolog, first produced comparable numbers of ERα+RFP+Erbb2+ and ERα-RFP-Erbb2+ MGECs. Early hyperplasia developed mostly from ERα+RFP+Erbb2+ cells and ERα-RFP-Erbb2+ cells in these lesions were rare. The subsequently developed ductal carcinomas in situ had 64% slow-proliferating ERα+RFP+Erbb2+ cells, 15% fast-proliferating ERα-RFP+Erbb2+ cells derived from ERα+RFP+Erbb2+ cells, and 20% fast-proliferating ERα-RFP-Erbb2+ cells. The advanced tumors had mostly ERα-RFP+Erbb2+ and ERα-RFP-Erbb2+ cells and only a very small population of ERα+RFP+Erbb2+ cells. In ERα-RFP+Erbb2+ cells, GATA3 and FoxA1 decreased expression and ERα promoter regions became methylated, consistent with the loss of ERα expression. Lung metastases consisted of mostly ERα-RFP+Erbb2+ cells, a few ERα-RFP-Erbb2+ cells, and no ERα+RFP+Erbb2+ cells. The high metastatic capacity of ERα-RFP+Erbb2+ cells was associated with ERK1/2 activation. These results show that the slow-proliferating, nonmetastatic ERα+RFP+Erbb2+ cells progressively lose ERα during tumorigenesis to become fast-proliferating, highly metastatic ERα-RFP+Erbb2+ cells. The ERα-Erbb2+ BrCs with an ERα+ origin are more aggressive than those ERα-Erbb2+ BrCs with an ERα- origin, and thus, they should be distinguished and treated differently in the future.

Airlift two-phase partitioning bioreactor for dichloromethane removal: Silicone rubber stimulated biodegradation and its auto-circulation.

Solid non-aqueous phases (NAPs), such as silicone rubber, have been used extensively to improve the removal of volatile organic compounds (VOCs). However, the removal of VOCs is difficult to be further improved because the poor understanding of the mass transfer and reaction processes. Further, the conventional reactors were either complicated or uneconomical. In view of this, herein, an airlift bioreactor with silicone rubber was designed and investigated for dichloromethane (DCM) treatment. The removal efficiency of Reactor 1 (with silicone rubber) was significantly higher than that of Reactor 2 (without silicone rubber), with corresponding higher chloride ion and CO2 production. It was found that Reactor 1 achieved a much better DCM shock tolerance capability and biomass stability than Reactor 2. Silicone rubber not only enhanced the mass transfer in terms of both gas/liquid and gas/microbial phases, but also decreased the toxicity of DCM to microorganisms. Noteworthily, despite the identical inoculum used, the relative abundance of potential DCM-degrading bacteria in Reactor 1 (91.2%) was much higher than that in Reactor 2 (24.3%) at 216 h. Additionally, the silicone rubber could be automatically circulated in the airlift bioreactor due to the driven effect of the airflow, resulting in a significant reduction of energy consumption.

Synthetic microbial consortia enable rapid assembly of pure translation machinery.

Assembly of recombinant multiprotein systems requires multiple culturing and purification steps that scale linearly with the number of constituent proteins. This problem is particularly pronounced in the preparation of the 34 proteins involved in transcription and translation systems, which are fundamental biochemistry tools for reconstitution of cellular pathways ex vivo. Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure. The expression of these proteins is controlled by synthetic genetic modules to produce the proteins at the correct ratios. We show that the pure multiprotein system is functional and reproducible, and has low protein contaminants. We also demonstrate its application in the screening of synthetic promoters and protease inhibitors. Our work establishes a novel strategy for producing pure translation machinery, which may be extended to the production of other multiprotein systems.

[Value of galactose-deficient IgA1 in the early diagnosis of Henoch-Schönlein purpura nephritis in children].

OBJECTIVE: To explore the value of galactose-deficient IgA1 (Gd-IgA1) in the early diagnosis of Henoch-Schönlein purpura nephritis (HSPN) in children. METHODS: A total of 67 hospitalized children who were definitely diagnosed with HSPN between January and April 2018 and 58 hospitalized children with Henoch-Schönlein purpura (HSP) were enrolled in the study. Twenty children undergoing routine physical examinations served as controls. The levels of serum and urine Gd-IgA1 were determined using ELISA. The receiver operating characteristic curve was used to analyze the value of serum Gd-IgA1 and urine Gd-IgA1/urine creatinine ratio in the diagnosis of HSPN. RESULTS: The level of serum Gd-IgA1 and urine Gd-IgA1/urine creatinine ratio in children with HSP or HSPN were significantly higher than those in healthy control group (P<0.01), with a significantly greater increase observed in children with HSPN (P<0.01). Serum Gd-IgA1 ≥1 485.57 U/mL and/or urine Gd-IgA1/urine creatinine ratio ≥105.74 were of favorable value in the diagnosis of HSPN. During the six-month follow-up of the 49 children with HSP, the incidence of HSPN was 47% (23/49), which included a 100% incidence in children with serum Gd-IgA1 ≥1 485.57 U/mL and a 73% incidence in children with urine Gd-IgA1/urine creatinine ratio ≥105.74. CONCLUSIONS: Serum and urine Gd-IgA1 is of favorable clinical value in the early diagnosis of HSPN.

Dynamics of the lipid droplet proteome of the Oleaginous yeast rhodosporidium toruloides.

Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.

Integrated omics study delineates the dynamics of lipid droplets in Rhodococcus opacus PD630.

Rhodococcus opacus strain PD630 (R. opacus PD630), is an oleaginous bacterium, and also is one of few prokaryotic organisms that contain lipid droplets (LDs). LD is an important organelle for lipid storage but also intercellular communication regarding energy metabolism, and yet is a poorly understood cellular organelle. To understand the dynamics of LD using a simple model organism, we conducted a series of comprehensive omics studies of R. opacus PD630 including complete genome, transcriptome and proteome analysis. The genome of R. opacus PD630 encodes 8947 genes that are significantly enriched in the lipid transport, synthesis and metabolic, indicating a super ability of carbon source biosynthesis and catabolism. The comparative transcriptome analysis from three culture conditions revealed the landscape of gene-altered expressions responsible for lipid accumulation. The LD proteomes further identified the proteins that mediate lipid synthesis, storage and other biological functions. Integrating these three omics uncovered 177 proteins that may be involved in lipid metabolism and LD dynamics. A LD structure-like protein LPD06283 was further verified to affect the LD morphology. Our omics studies provide not only a first integrated omics study of prokaryotic LD organelle, but also a systematic platform for facilitating further prokaryotic LD research and biofuel development.

[Comparison of therapeutic effects of prednisone combined with mycophenolate mofetil versus cyclosporin A in children with steroid-resistant nephrotic syndrome].

OBJECTIVE: To compare the therapeutic effects of prednisone combined with mycophenolate mofetil (MMF) versus cyclosporin A (CsA) in children with steroid-resistant nephrotic syndrome (SRNS). METHODS: The clinical data of 164 SRNS children who were treated with prednisone combined with MMF or CsA between January 2004 and December 2013 were collected, and the clinical effect of prednisone combined with MMF (MMF group, 112 children) or CsA (CsA group, 52 children) was analyzed retrospectively. RESULTS: At 1 month after treatment, the CsA group had a significantly higher remission rate than the MMF group (67.3% vs 42.9%; P<0.05). At 3 months after treatment, the CsA group also had a significantly higher remission rate than the MMF group (78.8% vs 63.3%; P<0.05). The 24-hour urinary protein excretion in both groups changed significantly with time (P<0.05) and differed significantly between the two groups (P<0.05). There were no serious adverse events in the two groups. CONCLUSIONS: Prednisone combined with MMF or CsA is effective and safe for the treatment of SRNS in children, and within 3 months of treatment, CsA has a better effect than MMF.

Mycophenolate mofetil therapy for steroid-resistant IgA nephropathy with the nephrotic syndrome in children.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) presents as nephrotic syndrome (NS) relatively rarely, and the current treatment experience of IgAN patients with NS is mostly with adults. The objective of our study was to investigate the efficacy of corticosteroids and mycophenolate mofetil (MMF) in treating childhood immunoglobulin A nephropathy (IgAN) with nephrotic syndrome. METHODS: A total of 58 children (39 boys and 19 girls) diagnosed with nephrotic syndrome and primary IgAN were enrolled in the study. All the patients were administered prednisone 2 mg/kg per day for 8 weeks. Steroid-resistant patients were treated with the combined use of MMF (dose of 20 ~ 30 mg/kg per day) and prednisone for 6-12 months. The prednisone dose was reduced stepwise during the combined treatment. RESULTS: Of the 58 children, 14 were steroid-sensitive (M, S, and T variants of the Oxford classification were 0 in most children), and 44 cases who presented serious pathological damage to the kidney were steroid-resistant. The estimated glomerular filtration rate (eGFR) of the steroid-resistant children (86.69 ± 26.85 ml/min/1.73 m(2)) was significantly lower (P < 0.05) than that of the steroid-sensitive children (106.89 ± 26.94 ml/min/1.73 m(2)). After 4 months of combined MMF treatment in 33 steroid-resistant children, complete remission of proteinuria was found in 21 cases, partial remission of proteinuria in 6 cases, and no response was found in 6 cases. Except for the T variant, other variants of the Oxford classification, including M, E, and S morphological variables, was not significantly different among patients complete remission, those with partial remission, and those with no response. The eGFR of children with complete remission of proteinuria (100.04 ± 18.47 ml/min/1.73 m(2)), that of those with partial remission (92.24 ± 27.63 ml/min/1.73 m(2)), and that of those with no response (72.17 ± 27.55 ml/min/1.73 m(2)) were significantly different (P < 0.05). CONCLUSION: Corticosteroid therapy showed satisfactory efficacy in IgAN children with nephrotic syndrome and slight pathological damage. The effect of MMF was good for steroid-resistant IgAN children, but poor for those with tubular atrophy/interstitial fibrosis and renal function impairment.

[Clinical characteristics of children with an initial onset of IgA nephropathy with nephrotic syndrome].

OBJECTIVE: To study the clinical characteristics of children with an initial onset of IgA nephropathy with nephrotic syndrome and compare them with children with primary nephrotic syndrome, in order to provide a theoretical basis for the differential diagnosis of the two diseases. METHODS: Fifty children diagnosed with an initial onset of IgA nephropathy with nephrotic syndrome were included in this study. Seventy-two children diagnosed with an initial onset of primary nephrotic syndrome served as the control group. The clinical and laboratory examination characteristics were compared between the two groups. RESULTS: The IgA nephropathy group had significantly higher incidence rates of gross haematuria, microscopic haematuria, hypertension, acute kidney injury, low serum high-density lipoprotein cholesterol, anemia, low serum complement C4, steroid resistance, and nephritis-type nephrotic syndrome and a significantly lower incidence of elevated serum IgE compared with the control group (P<0.05). There were significant differences in serum creatinine, serum uric acid, serum total cholesterol, serum high-density lipoprotein cholesterol, serum IgE, serum complement C4, and hemoglobin levels between the IgA nephropathy and the control groups (P<0.05). The thresholds of serum IgE (<131.2 IU/mL) and high-density lipoprotein cholesterol (<1.35 mmol/L) were reference parameters in the differential diagnosis of IgA nephropathy with nephrotic syndrome and primary nephrotic syndrome. CONCLUSIONS: Children with IgA nephropathy presenting nephrotic syndrome manifest mainly as nephritis type and steroid-resistant type in the clinical classification. Cinical manifestations accompanied by serum levels of high-density lipoprotein cholesterol and IgE are helpful for differential diagnosis of IgA nephropathy presenting nephrotic syndrome and primary nephrotic syndrome.

[Significance of trace deposition of immunoglobulin M in glomerular mesangium in children with minimal change nephrotic syndrome].

OBJECTIVE: To study the significance of trace immunoglobulin M (IgM) deposits in glomerular mesangium in children with minimal change primary nephrotic syndrome (PNS). METHODS: One hundred and six children who were clinically diagnosed with PNS and pathologically diagnosed with minimal change disease (MCD) and trace deposition of IgM in renal tissues were enrolled as subjects. Eighty-one PNS children with MCD but no deposition of immune complexes were used as the control group. The clinical characteristics and efficacies of glucocorticoids and immunosuppressants were retrospectively analyzed in the two groups. All patients were given full-dose prednisone by oral administration, and patients with glucocorticoid resistance or frequent relapses were additionally given immunosuppressants. RESULTS: The incidence of glucocorticoid resistance in the IgM deposit group was significantly higher than that in the control group (27.2% vs 12.3%; P<0.05). The incidence of frequent relapses in the IgM deposit group was also significantly higher than that in the control group (48.1% vs 10.4%; P<0.05). The complete remission rate for glucocorticoid-resistant patients treated with prednisone combined with mycophenolate mofetil (MMF) was 68% and 62% respectively in the IgM deposit and control groups (P>0.05). The relapse frequency in patients with frequent relapses was significantly reduced in both groups after treatment with prednisone and MMF in combination (P<0.05). CONCLUSIONS: Trace deposition of IgM in renal tissues may be an important factor for glucocorticoid resistance and frequent relapses in PNS children with MCD. Prednisone combined with MMF may be a better choice in the treatment of patients with glucocorticoid resistance or frequent relapses.

Clinical and pathological features of acute kidney injury in children.

OBJECTIVE: Based on the diagnostic and staging criteria of acute kidney injury (AKI), we analyze the clinical and pathological characteristics of children at different stages of AKI and explored their clinical significances. METHODS: 165 children with AKI were divided into stage 1, stage 2, and stage 3 groups. Clinical and pathologic characteristics of AKI children were analyzed. RESULTS: The three groups of patients showed significant differences in age, etiology, pathological damage, and the median recovery time of serum creatinine. Of the 165 patients, the incidence and duration of hematuria showed significant differences among the three groups, and the stage 1 group showed longer duration of proteinuria. CONCLUSION: The patients were largely in stage 1 and 3. The children with AKI in stage 1 were largely school-age children and acute glomerulonephritis (AGN) was the main etiology. The AKI children in stage 3 were mainly infants, of which the etiology was mainly drugs and septicemia. The pathological type was mainly acute tubulointerstitial nephritis, and the renal function recovery was slow.

Yeast perilipin Pet10p/Pln1p interacts with Erg6p in ergosterol metabolism.

Lipid droplets (LD) are highly dynamic organelles specialized for the regulation of energy storage and cellular homeostasis. LD consist of a neutral lipid core surrounded by a phospholipid monolayer membrane with embedded proteins, most of which are involved in lipid homeostasis. In this study, we focused on one of the major LD proteins, sterol C24-methyltransferase, encoded by ERG6. We found that the absence of Erg6p resulted in an increased accumulation of yeast perilipin Pet10p in LD, while the disruption of PET10 was accompanied by Erg6p LD over-accumulation. An observed reciprocal enrichment of Erg6p and Pet10p in pet10Δ and erg6Δ mutants in LD, respectively, was related to specific functional changes in the LD and was not due to regulation on the expression level. The involvement of Pet10p in neutral lipid homeostasis was observed in experiments that focused on the dynamics of neutral lipid mobilization as time-dependent changes in the triacylglycerols (TAG) and steryl esters (SE) content. We found that the kinetics of SE hydrolysis was reduced in erg6Δ cells and the mobilization of SE was completely lost in mutants that lacked both Erg6p and Pet10p. In addition, we observed that decreased levels of SE in erg6Δpet10Δ was linked to an overexpression of steryl ester hydrolase Yeh1p. Lipid analysis of erg6Δpet10Δ showed that PET10 deletion altered the composition of ergosterol intermediates which had accumulated in erg6Δ. In conclusion, yeast perilipin Pet10p functionally interacts with Erg6p during the metabolism of ergosterol.

Combining TIGIT Blockade with MDSC Inhibition Hinders Breast Cancer Bone Metastasis by Activating Antitumor Immunity.

Bone is the most common site of breast cancer metastasis. Bone metastasis is incurable and is associated with severe morbidity. Utilizing an immunocompetent mouse model of spontaneous breast cancer bone metastasis, we profiled the immune transcriptome of bone metastatic lesions and peripheral bone marrow at distinct metastatic stages, revealing dynamic changes during the metastatic process. We show that cross-talk between granulocytes and T cells is central to shaping an immunosuppressive microenvironment. Specifically, we identified the PD-1 and TIGIT signaling axes and the proinflammatory cytokine IL1ß as central players in the interactions between granulocytes and T cells. Targeting these pathways in vivo resulted in attenuated bone metastasis and improved survival, by reactivating antitumor immunity. Analysis of patient samples revealed that TIGIT and IL1ß are prominent in human bone metastasis. Our findings suggest that cotargeting immunosuppressive granulocytes and dysfunctional T cells may be a promising novel therapeutic strategy to inhibit bone metastasis. Significance: Temporal transcriptome profiling of the immune microenvironment in breast cancer bone metastasis revealed key communication pathways between dysfunctional T cells and myeloid derived suppressor cells. Cotargeting of TIGIT and IL1ß inhibited bone metastasis and improved survival. Validation in patient data implicated these targets as a novel promising approach to treat human bone metastasis.

Unbiased metastatic niche-labeling identifies estrogen receptor-positive macrophages as a barrier of T cell infiltration during bone colonization.

Microenvironment niches determine cellular fates of metastatic cancer cells. However, robust and unbiased approaches to identify niche components and their molecular profiles are lacking. We established Sortase A-Based Microenvironment Niche Tagging (SAMENT), which selectively labels cells encountered by cancer cells during metastatic colonization. SAMENT was applied to multiple cancer models colonizing the same organ and the same cancer to different organs. Common metastatic niche features include macrophage enrichment and T cell depletion. Macrophage niches are phenotypically diverse between different organs. In bone, macrophages express the estrogen receptor alpha (ERα) and exhibit active ERα signaling in male and female hosts. Conditional knockout of Esr1 in macrophages significantly retarded bone colonization by allowing T cell infiltration. ERα expression was also discovered in human bone metastases of both genders. Collectively, we identified a unique population of ERα+ macrophages in the metastatic niche and functionally tied ERα signaling in macrophages to T cell exclusion during metastatic colonization. HIGHLIGHTS: SAMENT is a robust metastatic niche-labeling approach amenable to single-cell omics.Metastatic niches are typically enriched with macrophages and depleted of T cells.Direct interaction with cancer cells induces ERα expression in niche macrophages. Knockout of Esr1 in macrophages allows T cell infiltration and retards bone colonization.

[Clinical features and treatment of acute clenbuterol poisoning in children].

OBJECTIVE: To study clinical features, treatment and curative effects in children with acute clenbuterol poisoning, in order to provide a basis for early diagnosis and treatment. METHODS: Clinical data of 28 hospitalized children with acute clenbuterol poisoning in April 2011 were retrospectively studied. RESULTS: Of the 28 patients, there were 15 males and 13 females, aged 1 to 13 years (mean age 6.5±4.8 years). Vomiting, palpitations and limb shaking were found as main clinical manifestations in the patients. Main changes of blood biochemical included hypokalemia, lactic acidosis, hyperglycemia, hypsocreatinkinase. Snus tachycardia and S-T segment depression were observed on ECG. Patients' symptoms were gradually alleviated after 12-78 hours by use of beta blockers, potassium supplement, protecting the heart and other symptomatic and supportive treatment. Blood biochemical indexes were improved after 48 hours of admission. All of the patients were cured after 5 days. The symptoms of the patients do not longer occur during a follow up of half a month. CONCLUSIONS: Acute clenbuterol poisoning is characterized by vomiting, palpitations, limb shaking, hypokalemia, lactic acidosis and tachycardia in children. An early effective treatment of this disease can improve prognosis in children.

Pilose antler (Cervus elaphus Linnaeus) polysaccharide and polypeptide extract inhibits bone resorption in high turnover type osteoporosis by stimulating the MAKP and MMP-9 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Pilose antler is a traditional Chinese medicine used to improve kidney function, strengthen tendons and bones, and prolong life, among other uses. It is widely employed in the treatment of osteoporosis. However, the molecular mechanisms underlying the treatment of high turnover osteoporosis are not fully understood. AIM OF THE STUDY: The present study aimed to investigate the molecular mechanism underlying pilose antler polysaccharide and polypeptide extracts in inhibiting bone resorption in high turnover osteoporosis, and compare the effects of the two components alone and in combination to explore whether they could produce synergistic enhancement effects. MATERIALS AND METHODS: The quantitative and qualitative characteristics of pilose antler polysaccharide and polypeptide extracts were detected by UV-visible spectrophotometry and high-performance liquid chromatography. A rat model of retinoic acid-induced osteoporosis was used to evaluate the inhibitory effect of the extracts on bone resorption. Enzyme-linked immunosorbent assay (ELISA) was used to detect the activity of factors related to high turnover type osteoporosis in rat serum. Western blotting was used to detect the expression of proteins related to the MAKP and MMP-9 signaling pathways in rat femurs. Fluorescence quantitative PCR was used to detect the transcription levels of genes related to the MAKP and MMP-9 signaling pathways in rat femur tissues. Hematoxylin and eosin staining were used to observe the osteoprotective effects of pilose antler polysaccharides and polypeptides. RESULTS: The yield of pilose antler polysaccharides was 8.3%, and was mainly composed of mannose, glucosamine hydrochloride, glucuronic acid, Galacturonic acid, Galactose hydrochloride, glucose, and galactose. The yield of the polypeptides was 26.2%, and eighty percent of the molecular weight of the antler polypeptides was 1.6 kDa-7kD, among which, the molecular weight of 7kD peptide accounted for 52% of the total. Both polysaccharides and peptides could reduce the activities of TRACP, OCN, ERK1, JNK, and MMP-9 in rat serum and reduce both the protein expression and gene transcription levels of ERK1, JNK, and MMP-9 in rat femur tissue with significant differences compared with the model group. Both extracts exerted significant protective effects on rat femur tissue. The effect of pilose antler polypeptides alone was better than that of polysaccharides either alone or in combination. CONCLUSIONS: Pilose antler polysaccharides, polypeptides, and their mixtures could inhibit the occurrence of bone resorption of high turnover osteoporosis by stimulating the MAKP and MMP-9 signaling pathways to reduce the expression of the ERK1, JNK, and MMP-9 genes and proteins, and could help alleviate bone loss caused by retinoic acid. Pilose antler polypeptides had a stronger effect on inhibiting bone resorption. The combination of the two components did not show synergistic enhancement effect, and the polysaccharide tended to moderate the inhibitory enhancement effect of the polypeptide.

Rational Protein Design Yields a CD20 CAR with Superior Antitumor Efficacy Compared with CD19 CAR.

Chimeric antigen receptors (CAR) are fusion proteins whose functional domains are often connected in a plug-and-play manner to generate multiple CAR variants. However, CARs with highly similar sequences can exhibit dramatic differences in function. Thus, approaches to rationally optimize CAR proteins are critical to the development of effective CAR T-cell therapies. Here, we report that as few as two amino-acid changes in nonsignaling domains of a CAR were able to significantly enhance in vivo antitumor efficacy. We demonstrate juxtamembrane alanine insertion and single-chain variable fragment sequence hybridization as two strategies that could be combined to maximize CAR functionality, and describe a CD20 CAR that outperformed the CD19 CAR in antitumor efficacy in preclinical in vitro and in vivo assays. Precise changes in the CAR sequence drove dramatically different transcriptomic profiles upon antigen stimulation, with the most efficacious CAR inducing an enrichment in highly functional memory T cells upon antigen stimulation. These findings underscore the importance of sequence-level optimization to CAR T-cell function, and the protein-engineering strategy described here may be applied to the development of additional CARs against diverse antigens. See related Spotlight by Scheller and Hudecek, p. 142.

Identifying molecular and functional similarities and differences between human primary cardiac valve interstitial cells and ventricular fibroblasts.

Introduction: Fibroblasts are mesenchymal cells that predominantly produce and maintain the extracellular matrix (ECM) and are critical mediators of injury response. In the heart, valve interstitial cells (VICs) are a population of fibroblasts responsible for maintaining the structure and function of heart valves. These cells are regionally distinct from myocardial fibroblasts, including left ventricular cardiac fibroblasts (LVCFBs), which are located in the myocardium in close vicinity to cardiomyocytes. Here, we hypothesize these subpopulations of fibroblasts are transcriptionally and functionally distinct. Methods: To compare these fibroblast subtypes, we collected patient-matched samples of human primary VICs and LVCFBs and performed bulk RNA sequencing, extracellular matrix profiling, and functional contraction and calcification assays. Results: Here, we identified combined expression of SUSD2 on a protein-level, and MEOX2, EBF2 and RHOU at a transcript-level to be differentially expressed in VICs compared to LVCFBs and demonstrated that expression of these genes can be used to distinguish between the two subpopulations. We found both VICs and LVCFBs expressed similar activation and contraction potential in vitro, but VICs showed an increase in ALP activity when activated and higher expression in matricellular proteins, including cartilage oligomeric protein and alpha 2-Heremans-Schmid glycoprotein, both of which are reported to be linked to calcification, compared to LVCFBs. Conclusion: These comparative transcriptomic, proteomic, and functional studies shed novel insight into the similarities and differences between valve interstitial cells and left ventricular cardiac fibroblasts and will aid in understanding region-specific cardiac pathologies, distinguishing between primary subpopulations of fibroblasts, and generating region-specific stem-cell derived cardiac fibroblasts.

Bone Metastasis Initiation Is Coupled with Bone Remodeling through Osteogenic Differentiation of NG2+ Cells.

The bone microenvironment is dynamic and undergoes remodeling in normal and pathologic conditions. Whether such remodeling affects disseminated tumor cells (DTC) and bone metastasis remains poorly understood. Here, we demonstrated that pathologic fractures increase metastatic colonization around the injury. NG2+ cells are a common participant in bone metastasis initiation and bone remodeling in both homeostatic and fractured conditions. NG2+ bone mesenchymal stem/stromal cells (BMSC) often colocalize with DTCs in the perivascular niche. Both DTCs and NG2+ BMSCs are recruited to remodeling sites. Ablation of NG2+ lineage impaired bone remodeling and concurrently diminished metastatic colonization. In cocultures, NG2+ BMSCs, especially when undergoing osteodifferentiation, enhanced cancer cell proliferation and migration. Knockout of N-cadherin in NG2+ cells abolished these effects in vitro and phenocopied NG2+ lineage depletion in vivo. These findings uncover dual roles of NG2+ cells in metastasis and remodeling and indicate that osteodifferentiation of BMSCs promotes metastasis initiation via N-cadherin-mediated cell-cell interaction. SIGNIFICANCE: The bone colonization of cancer cells occurs in an environment that undergoes constant remodeling. Our study provides mechanistic insights into how bone homeostasis and pathologic repair lead to the outgrowth of disseminated cancer cells, thereby opening new directions for further etiologic and epidemiologic studies of tumor recurrences. This article is highlighted in the In This Issue feature, p. 247.

Osteoprogenitor-GMP crosstalk underpins solid tumor-induced systemic immunosuppression and persists after tumor removal.

Remote tumors disrupt the bone marrow (BM) ecosystem (BME), eliciting the overproduction of BM-derived immunosuppressive cells. However, the underlying mechanisms remain poorly understood. Herein, we characterized breast and lung cancer-induced BME shifts pre- and post-tumor removal. Remote tumors progressively lead to osteoprogenitor (OP) expansion, hematopoietic stem cell dislocation, and CD41- granulocyte-monocyte progenitor (GMP) aggregation. The tumor-entrained BME is characterized by co-localization between CD41- GMPs and OPs. OP ablation abolishes this effect and diminishes abnormal myeloid overproduction. Mechanistically, HTRA1 carried by tumor-derived small extracellular vesicles upregulates MMP-13 in OPs, which in turn induces the alterations in the hematopoietic program. Importantly, these effects persist post-surgery and continue to impair anti-tumor immunity. Conditional knockout or inhibition of MMP-13 accelerates immune reinstatement and restores the efficacies of immunotherapies. Therefore, tumor-induced systemic effects are initiated by OP-GMP crosstalk that outlasts tumor burden, and additional treatment is required to reverse these effects for optimal therapeutic efficacy.