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Supplementary figure 1. (A) Immunolabeling for phosphorylated p63 (p-p63), CK5, CK20 and Ki67 in forestomach sections of approximately 6-week-old mice with the listed genotypes. Supplementary figure 2. Individual channels microscopy imaging for the data presented in Figure 2F. Supplementary figure 3. (A) RT-PCR electrophoretic product for the listed genes in the forestomach fibroblasts from approximately 6-week-old mice with the listed genotypes. Supplementary figure 4. Supplementary figure 5. Uncropped western blots

Supplementary methods and figure legends

Tumor vessel co-option (VCO) is a non-angiogenic vascularization mechanism that is a possible cause of resistance to anti-angiogenic therapy (AAT). Multiple tumors are hypothesized to primarily rely on growth factor signaling-induced sprouting angiogenesis, which is often inhibited during AAT. During VCO however, tumors invade healthy tissues by hijacking pre-existing blood vessels of the host organ to secure their blood and nutrient supply. Although VCO has been described in the context of AAT resistance, the molecular mechanisms underlying this process and the profile and characteristics of co-opted vascular cell types (endothelial cells (ECs) and pericytes) remain poorly understood, resulting in the lack of therapeutic strategies to inhibit VCO (and to overcome AAT resistance). In the past few years, novel next-generation technologies (such as single-cell RNA sequencing) have emerged and revolutionized the way of analyzing and understanding cancer biology. While most studies utilizing single-cell RNA sequencing with focus on cancer vascularization have centered around ECs during sprouting angiogenesis, we propose that this and other novel technologies can be used in future investigations to shed light on tumor EC biology during VCO. In this review, we summarize the molecular mechanisms driving VCO known to date and introduce the models used to study this phenomenon to date. We highlight VCO studies that recently emerged using sequencing approaches and propose how these and other novel state-of-the-art methods can be used in the future to further explore ECs and other cell types in the VCO process and to identify potential vulnerabilities in tumors relying on VCO. A better understanding of VCO by using novel approaches could provide new answers to the many open questions, and thus pave the way to develop new strategies to control and target tumor vascularization. ispartof: FRONTIERS IN ONCOLOGY vol:12 ispartof: location:Switzerland status: published

Skin wound repair is essential for organismal survival and failure of which leads to non‐healing wounds, a leading health issue worldwide. However, mechanistic understanding of chronic wounds remains a major challenge due to lack of appropriate genetic mouse models. αSMA(+) myofibroblasts, a unique class of dermal fibroblasts, are associated with cutaneous wound healing but their precise function remains unknown. We demonstrate that genetic depletion of αSMA(+) myofibroblasts leads to pleiotropic wound healing defects, including lack of reepithelialization and granulation, dampened angiogenesis, and heightened hypoxia, hallmarks of chronic non‐healing wounds. Other wound‐associated FAP(+) and FSP1(+) fibroblasts do not exhibit such dominant functions. While type I collagen (COL1) expressing cells play a role in the repair process, COL1 produced by αSMA(+) myofibroblasts is surprisingly dispensable for wound repair. In contrast, we show that β1 integrin from αSMA(+) myofibroblasts, but not TGFβRII, is essential for wound healing, facilitating contractility, reepithelization, and vascularization. Collectively, our study provides evidence for the functions of myofibroblasts in β1 integrin‐mediated wound repair with potential implications for treating chronic non‐healing wounds.

The vascular endothelium is highly heterogeneous, with phenotypically and functionally distinct endothelial cell (EC) subtypes, partially driven by distinct metabolic programs. While this heterogeneity is important to maintain the multiple functions of the endothelium during tissue repair, it also contributes to challenges to therapeutically modulate ECs in pathological contexts. While the EC heterogeneity is far from being entirely unraveled, recent advances in single-cell technologies have greatly contributed to the identification of different EC subsets and cell states. In this chapter, we discuss new EC subsets captured by single-cell studies in health and disease, also focusing on metabolic EC signatures. We also highlight how such metabolic signatures could be exploited in therapeutic settings to promote or block angiogenesis.

Endothelial cells (ECs) constitute the inner lining of vascular beds in mammals and are crucial for homeostatic regulation of blood vessel physiology, but also play a key role in pathogenesis of many diseases, thereby representing realistic therapeutic targets. However, it has become evident that ECs are heterogeneous, encompassing several subtypes with distinct functions, which makes EC targeting and modulation in diseases challenging. The rise of the new single-cell era has led to an emergence of studies aimed at interrogating transcriptome diversity along the vascular tree, and has revolutionized our understanding of EC heterogeneity from both a physiological and pathophysiological context. Here, we discuss recent landmark studies aimed at teasing apart the heterogeneous nature of ECs. We cover driving (epi)genetic, transcriptomic, and metabolic forces underlying EC heterogeneity in health and disease, as well as current strategies used to combat disease-enriched EC phenotypes, and propose strategies to transcend largely descriptive heterogeneity towards prioritization and functional validation of therapeutically targetable drivers of EC diversity. Lastly, we provide an overview of the most recent advances and hurdles in single EC OMICs. ispartof: CARDIOVASCULAR RESEARCH vol:119 issue:1 pages:6-27 ispartof: location:England status: published

Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent. ispartof: CELL REPORTS vol:35 issue:11 ispartof: location:United States status: published

The vascular endothelium is characterized by a remarkable level of plasticity, which is the driving force not only of physiological repair/remodeling of adult tissues but also of pathological angiogenesis. The resulting heterogeneity of endothelial cells (ECs) makes targeting the endothelium challenging, no less because many EC phenotypes are yet to be identified and functionally inventorized. Efforts to map the vasculature at the single-cell level have been instrumental to capture the diversity of EC types and states at a remarkable depth in both normal and pathological states. Here, we discuss new EC subtypes and functions emerging from recent single-cell studies in health and disease. Interestingly, such studies revealed distinct metabolic gene signatures in different EC phenotypes, which deserve further consideration for therapy. We highlight how this metabolic targeting strategy could potentially be used to promote (for tissue repair) or block (in tumor) angiogenesis in a tissue or even vascular bed-specific manner. ispartof: ANGIOGENESIS vol:24 issue:2 pages:311-326 ispartof: location:Germany status: published

Endothelial cell migration is indispensable for (tumour) angiogenesis and is fuelled by glycolysis. The metabolic underpinnings have been only partially unravelled to date, partly because of the lack of appropriate tools to analyse metabolic flux at the single-cell level. In this issue, Wu et al. introduce a novel imaging assay to capture glycolytic flux during motion in single endothelial cells. With this new tool, the authors uncovered a glycolysis-driven cytoskeletal remodelling apparatus that propels endothelial cell motility.

Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies against the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological ELISA using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 reverse transcription PCR–confirmed, SARS-CoV-2–hospitalized patients, as well as 464 healthy and non–COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies against the 2 different viral proteins showed a moderate correlation. Antibodies against N-protein were detected at a rate of 3.6% in healthy and non–COVID-19 sera collected during the pandemic in 2020, whereas 1.9% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD–binding antibodies exhibited neutralizing capacity, but only 74% of N-protein–positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein–binding antibodies does not always correlate with presence of S-RBD–neutralizing antibodies and caution against the extensive use of N-protein–based serology testing for determination of potential COVID-19 immunity., Detection of SARS-CoV-2 nucleocapsid protein binding antibodies does not always correlate with the presence of spike protein receptor binding domain neutralizing antibodies.

Deregulated transforming growth factor-β (TGFβ) signaling is a common feature of many epithelial cancers. Deletion of TGFβ receptor type 2 (TGFBR2) in fibroblast specific protein-1 (FSP1)-positive stromal cells induces squamous cell carcinoma in the murine forestomach, implicating fibroblast-derived hepatocyte growth factor (HGF) as the major driver of the epithelium carcinogenesis. Prior to cancer development, hyperproliferative FSP1+ fibroblasts lacking TGFBR2 accumulate in the forestomach, disrupting the regulatory signaling cross-talk with the forestomach epithelium. Here, concurrent loss in TGFBR2 and SMAD4 completely abrogates the development of forestomach cancer. Bone morphogenic protein-7 (BMP7) was highly upregulated in forestomach cancer tissue, activating Smad1/5/8 signaling, cell proliferation, and HGF production in TGFBR2-deficient FSP1+ fibroblasts. This stimulation by BMP7 was lost in the combined TGFBR2 and SMAD4 double knockout fibroblasts, which included a profound decrease in HGF expression. Thus, Smad4-mediated signaling is required to initiate epithelial carcinogenesis subsequent to TGFBR2 deletion in FSP1+ fibroblasts. Implications: These findings reveal a complex cross-talk between epithelial cells and the stroma, wherein Smad4 is required to elicit squamous cell carcinomas in the forestomach of mice with TGFBR2-deficient stromal cells. Mol Cancer Res; 16(10); 1568–78. ©2018 AACR.

Cathepsin G is broadly expressed in acute myeloid leukemia and is an effective immunotherapeutic target

On endothelial cells, the TGFβ/BMP-9 coreceptor, endoglin, emerged as a promising antiangiogenic target in colorectal cancer. Its promiscuous expression by cancer-associated fibroblasts (CAF) emerges to facilitate metastasis. The multitarget impact of neutralizing endoglin may offer added benefit in controlling colorectal cancer. See related article by Paauwe et al., p. 6331

Deregulated transforming growth factor-β (TGFβ) signaling is a common feature of many epithelial cancers. Deletion of TGFβ receptor type 2 (TGFBR2) in fibroblast specific protein-1 (FSP1) positive stromal cells induces squamous cell carcinoma in the murine forestomach, implicating fibroblast-derived hepatocyte growth factor (HGF) as the major driver of the epithelium carcinogenesis. Prior to cancer development, hyperproliferative FSP1(+) fibroblasts lacking TGFBR2 accumulate in the forestomach, disrupting the regulatory signaling crosstalk with the forestomach epithelium. Here, concurrent loss in TGFBR2 and SMAD4 completely abrogates the development of forestomach cancer. Bone morphogenic protein-7 (BMP7) was highly upregulated in forestomach cancer tissue, activating Smad1/5/8 signaling, cell proliferation and HGF production in TGFBR2-deficient FSP1(+) fibroblasts. This stimulation by BMP7 was lost in the combined TGFBR2 and SMAD4 double knockout fibroblasts, which included a profound decrease in HGF expression. Thus, Smad4-mediated signaling is required to initiate epithelial carcinogenesis subsequent to TGFBR2 deletion in FSP1(+) fibroblasts.

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.

Introduction. Targeting antigen-driven B-cell receptor (BCR) signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), but not germinal center B-cell (GCB) DLBCL. We have formally confirmed that GCB-DLBCL cell lines utilize tonic BCR signaling, by showing: 1) sensitivity (variable) to knockout (KO) of the BCR, SYK, and CD19; 2) dependence on CD79A ITAM phosphorylation; and 3) independence from BCR antigen specificity. However, uncertainty remains about molecular events in upstream parts of tonic BCR signaling, why dependence of GCB-DLBCL cells on tonic BCR signaling is variable, and their clinical relevance. Methods. We used CRISPR/Cas9 methods to modify selected genes by KO and/or knock-in (KI) of the cDNA of a fluorescent protein (FP; e.g., GFP), with the FP serving as a marker of cells with gene KO or modification, or as a gene-fused tag for localization or quantitation. Cells expressing a membrane-targeted Forster resonance energy transfer (FRET) based AKT activity reporter (Lyn-AktAR2) were used to measure AKT activity directly by flow cytometry (FCM). Results. The effect of KI of CD79A Y188F mutation alone was similar to complete BCR KO, implying that CD79A Y188 phosphorylation is essential for tonic BCR signal transduction. Western blot analysis of GCB-DLBCL cell lines after BCR KO showed variable decreases of AKT S473 phosphorylation (frequently used as surrogate measure of AKT activity), but these did not correlate well with the variable decreases in proliferation of GCB-DLBCL cell lines caused by BCR KO. Measuring AKT activity directly (Fig. 1), or by another indirect approach (surface expression of CXCR4, a target gene of FOXO1 inhibited by AKT activity), showed high correlation between decreases in AKT activity and proliferation after BCR KO. In contrast to the variable effect of BCR KO on growth, pan-AKT KO was uniformly growth-slowing in GCB-DLBCL lines (Fig. 2). Interestingly, baseline surface density of BCR units in GCB lines, quantified by FCM using CD79A-GFP KI cells or anti-CD79B staining, correlated highly with reduction in growth or AKT activity caused by BCR KO (Fig. 3). These findings lead us to conclude that the BCR contributes to AKT activation in GCB-DLBCL cell lines, to a variable degree determined by BCR surface density. We also conclude that BCR surface density is determined by cell line-specific factors, as well as immunoglobulin heavy (IgH) and light (IgL) hypervariable region (HVR) sequences, based on measurements of BCR surface levels after exchanging endogenous HVR sequences in OCI-Ly19 and OCI-Ly7 cell lines for HVRs derived from other GCB and ABC-DLBCL cell lines. Reduction of AKT activity after BCR KO (measured by FRET reporter) and baseline BCR surface density in GCB-DLBCL cell lines also correlated well with the sensitivity of GCB-DLBCL lines to the clinically-tested SYK inhibitor (P505-15, PRT062607) or FDA-approved PI3K p110d isoform specific inhibitor (idelalisib). Interestingly, isogenic GCB-DLBCL cell lines with KO of PTEN, a negative regulator of AKT activation, were substantially more resistant to both inhibitors. A crucial role of PTEN deletion in overcoming dependence on tonic BCR signaling in GCB-DLBCL is supported by evidence from two naturally PTEN-deficient cell lines: SUDHL10, which adjusts to BCR KO and resumes normal growth, and HT, which lacks BCR expression, due to a frameshifting deletion in its IgH HVR. Re-expression of the BCR in HT, by KI to correct the IgH sequence, does not affect HT cell line growth. Conclusion. Our findings suggest a biomarker-guided therapeutic strategy in GCB-DLBCL: targeting tonic BCR signaling in BCR-high patients, by inhibiting CD79A phosphorylation, SYK, or PI3K, and downstream targeting of AKT in BCR-low and/or PTEN-deficient patients. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Disclosures Burger: Pharmacyclics: Research Funding. Westin:Chugai: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; ProNAi: Membership on an entity's Board of Directors or advisory committees.

Vascularized fibular transplantation has found wide application for long bone reconstruction in both congenital and acquired defects. However, in children the growth of these transplants is limited and requires the epiphysis if it is to be substantial. Also, the reconstruction of a joint is best achieved with autologus hyaline cartilage. Vascularized fibular epiphyseal transplantation, thus, may play a role in limb salvage procedures in children to both preserve joint function and facilitate long bone growth. The present paper discusses the history of vascularized epiphyseal transplantation both in the laboratory and clinical realm. In discussing its advantages and associated problems, special emphasis is directed at the vascular factors, the endocrine environment, the innervation, mechanical stressors, the recipient site and the donor site. Two case examples are presented to demonstrate the technique, the results and the long term outcome.

Introduction. Targeting BCR signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), but not the germinal center B-cell (GCB) subtype. Active BCR signaling in GCB-DLBCL was suggested by studies with a Syk inhibitor and our previous studies using BCR knockout (KO). We addressed these questions: why is the BCR active in DLBCL, and how does it signal in GCB-DLBCL? Methods. We used CRISPR/Cas9 technology to modify selected genes by KO or homologous recombination-mediated knock-in (KI). For some genes KI was used to express a fluorescent protein (FP; e.g., GFP) instead of the targeted gene (KI/KO), or to modify the targeted gene together with KI of an FP, for detection of modified cells. Results. In GCB lines (OCI-Ly7 and OCI-Ly19) and ABC lines (U2932 and HBL-1), we simultaneously replaced the hypervariable region (HVR) exons of both immunoglobulin heavy (IgH) and light chains (IgL) with HVR sequences from normal B cells recognizing tetanus toxoid (TT). GFP and CFP respectively marked KI of IgH and IgL HVRs, and KI of the endogenous HVR sequences in each line served as controls. In CFP+/GFP+ cells, the TT specific BCR (TT-BCR) was expressed at similar or higher levels than the endogenous BCR (endo-BCR) and was functional, as shown by calcium flux in response to TT. The TT-BCR maintained growth of GCB lines (Fig. 1), indicating that they use "tonic", antigen-independent BCR signaling. Other features of tonic signaling were confirmed in more GCB lines: 1) the toxicity of BCR KO, which eliminates AKT S473 phosphorylation, was rescued by PTEN KO or expression of constitutively active AKT (mAKT), showing that BCR signaling serves principally to activate PI3K/AKT; and 2) KO of SYK or CD19, or truncation or ITAM mutation of the cytoplasmic tail of CD79A, none of which affect surface BCR levels, were as toxic as BCR KO but were non-toxic in BCR/PTEN double-KO cells. In contrast, the TT-BCR was as growth-slowing as BCR KO to the ABC line U2932 (Fig. 1), and substantially toxic to HBL-1, indicating that BCR signaling is self antigen-dependent in ABC-DLBCL. Reversion of somatic hypermutations in the U2932 HVRs was also as growth-slowing as BCR KO (Fig. 1), suggesting that self-antigen reactivity developed during BCR affinity maturation. Tonic signaling by the TT-BCR provided a detectable benefit (as compared to BCR KO) in PTEN-expressing HBL-1, whereas there was no difference between TT-HVR BCR and BCR KO in PTEN-deficient U2932. The surface TT-BCR level was higher than the endo-BCR level in ABC lines, and dropped with TT stimulation, suggesting that endo-BCRs in ABC lines undergo constant antigen stimulation with BCR internalization. The presumed self-antigen in ABC lines seems to be cell line-specific, since HVRs from ABC lines TMD8 and HBL-1 did not rescue growth of U2932. BCR KO in ABC lines was also not rescued by PTEN KO or mAKT. In cells whose BCRs were labeled by KI to fuse GFP to CD79A, super-resolution microscopy showed macro-clustering of BCR complexes at the surface of ABC line HBL-1, not seen in GCB lines (Fig. 2). Several findings suggested the clinical potential of targeting tonic BCR signaling in DLBCL: 1) clinical trial-stage inhibitors of SYK (P505-15) and PI3K (idelalisib) were toxic to GCB lines (less so with PTEN KO); 2) GCB lines (6/8) were sensitized by BCR KO to an in vitro CHOP-like regimen; 3) P505-15 or idelalisib sensitized GCB lines (3/3) to CHOP in vitro; and 4) evidence of tonic signaling in ABC line HBL-1 after removing antigen-driven signaling by HVR replacement. Conclusion. The BCR provides antigen-independent tonic signals to activate PI3K/AKT in GCB-DLBCL and antigen-dependent signaling in ABC-DLBCL. Targeting of B-cell specific tonic signling alone or in combination could be clinically effective in both types of DLBCL. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Disclosures Westin: Spectrum: Research Funding.

ispartof: CIRCULATION vol:145 issue:20 pages:1503-1505 ispartof: location:United States status: published