Luciferase-labeled ID8-Luc mouse ovarian cancer cells (1.0106 cells) were inoculated intraperitoneally into age-matched (4 to 6 weeks old), female C57BL/6 (Taconic Biosciences), and CD36flox/floxTie2Cre mice. 1000nM, min conc. Data represented as mean valuesSD determined by the ordinary one-way ANOVA Tukeys multiple comparisons test; n=5 for both C & D; for D; n=4 for E and F. To determine the potential clinical relevance of CD5L overexpression, we first interrogated a select cohort of ovarian cancer patients identified as bevacizumab responders versus non-responders. Circulating AIM prevents hepatocellular carcinoma through complement activation. 3B). This was followed by washing and incubation at 37C with alkaline phosphatase conjugated goat anti-human IgG (H+L) secondary antibody (Pierce/Thermo Scientific, Cat. We observed almost no expression of CD5L in any of these populations, with only a few monocytes and B cells showing some level of expression (each dot represents one single cell; Fig. The unbound sequences were incubated with the target protein CD5L (selection step) at room temperature. After 48h at 37C, 5% CO2, the cells were treated with either 40g/mL of R-35 or 200ng/ml of rCD5L for 6h. The cells were washed twice with PBS and serum-deprived for 1h before adding 100L TF2-C12 Fatty Acid Stock Solution (Sigma-Aldrich, MAK156). J. Pathol. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGFVEGFR1 interaction. : Data curation and formal analysis; S.P. 1EG). Tumor progression was monitored by weekly bioluminescence. Indeed, S63 and S72 sequences had no or weak binding for CD5L, whereas S47 was able to bind VEGF (used as a negative control), and CD5L target protein (TableS1). Nat. The solid phase panning method was used for the selection of CD5L binders by ELISA21. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. To examine potential mechanisms underlying resistance to anti-VEGF antibody (AVA) therapy, we used mouse models to identify tumors that demonstrated growth subsequent to a period of initial responseto treatment. S16AE). Treatment 2: RF24 cells were treated with 200ng of CD5L protein for 2h, followed by LY294002 (50M) for 6h, and then added to each well. Many antiangiogenic drugs, particularly those targeting the VEGF/VEGFR pathway, have been developed and are approved for cancer treatment. Genes were excluded if the percentile of the log-ratio variation was <50%, the percentage of data missing or filtered out exceeded 50%, or the percentage of absent (i.e., detection call = A) data exceeded 50%. Introduction The role of VEGF in driving tumor angiogenesis has made it an attractive target for therapeutic interventions, being bevacizumab, an FDA-approved humanized monoclonal antibody against VEGF, the most promising of them ( 1 ). Current nAMD standard of care dictates frequent intravitreal (IVT) anti-vascular endothelial growth factor (VEGF) injections, which places a substantial burden on patients, caregivers, and physicians. 1D). After transfection of 48h with hCD5L siRNAs or after treatment with either CD5L aptamer or anti-CD5L antibody (40g/ml), RF24 cells (1.0105) in MEM serum-free medium with CD5L protein (200ng/ml) were seeded into the upper chamber of the transwell. From a conceptual perspective, we concluded that hypoxia incurred due to prolonged VEGF blockade ultimately drives the overexpression of CD5L through the upregulation of transcription factor PPARG. Nature Communications (Nat Commun) We examined the genomic profiles of tumor-associated endothelial cells collected at pretreatment, at the maximal response, and at tumor progression and found substantially elevated CD5L levels at the time of progression. Tissue specimens were either fixed by using 10% buffered formalin, frozen in OCT (Miles, Inc., Elkhart, IN), or snap-frozen in liquid nitrogen. : Conceptualization, resources, and supervision; V.d.F. Biol. The addition of the R-35 antibody to HUVEC spheroids treated with CD5L protein blocked the observed increase in sprout length in HUVEC spheroids induced by the control antibody plus CD5L protein (Fig. In brief, cross-linked cells were collected, lysed, sonicated, and subsequently subjected to immunoprecipitation with PPARG (Cell Signaling) antibody or IgG control. A number of molecules involved in angiogenesis have. Mol. 2L). To quantify mean vessel density in the mouse tumor samples, the number of blood vessels that stained positive for CD31 was recorded in five random 0.159-mm2 fields at 200 magnification. Understanding and overcoming adaptive changes to anti-VEGF drugs represent an opportunity to further enhance the efficacy of these drugs and potentially delay or prevent adaptive resistance3,4,5,6. Since CD36 has been previously reported to be one of the major receptors for CD5L in macrophages7,8, we sought to determine the effect of exogenous CD5L treatment on CD36 expression. Cell Rep. 9, 6174 (2014). Human immortalized umbilical endothelial cells (RF24) were grown in MEM medium containing supplements (nonessential amino acids, sodium pyruvate, MEM vitamins, and glutamine; Life Technologies, Grand Island, NY). To determine whether PPARG may serve as an upstream regulator of CD5L, we ectopically expressed PPARG in RF24 endothelial cells. Antiangiogenesis represents a validated therapeutic strategy for a variety of disorders. Vatalanib, a small-molecule anticancer drug that inhibits angiogenesis (Fig. Scale bar=100m. Tie2-cre transgene and floxed PPARG alleles were distinguished by PCR by using primers (Supplementary Table3). Antibodies were purified with the use of A/G affinity resin to purity >95% as reported previously23. PubMedGoogle Scholar. Gynecol. Lung Cell. Approval for both sets of human samples was provided by the Institutional Review Board (IRB). We also tested the effect of the R-35 antibody on primary endothelial cells. Peer reviewer reports are available. CAS A Time point at which SKOV3ip1 ovarian cancer tumors were isolated during the course of B20 treatment. Recent studies have investigated the role of antiangiogenic agents in antitumor immunity, especially in recent 3 years. Macrophage-derived AIM is endocytosed into adipocytes and decreases lipid droplets via inhibition of fatty acid synthase activity. KaplanMeier survival curves were generated and compared with the use of log-rank statistics to assess the effect of tumor vascular CD5L expression on human overall survival and to determine survival in the Tie2-cre;PPARG KO mouse model. N. Engl. CD5L expression levels were determined with the use of a CD5L ELISA Kit (MyBioSource, San Diego, CA) according to the manufacturers recommendations for both cell culture media as well as human serum samples. We found that both PPARG and CD5L mRNA expression levels were significantly higher after 30h of CoCl2 incubation than after 6h as well as non-treated cells, further validating that hypoxia-like conditions lead to increased PPARG and CD5L expression (Fig. By submitting a comment you agree to abide by our Terms and Community Guidelines. Single B cells were isolated and screened for CD5L-binding antibodies by using ELISA for initial positive hits. 5A). was supported by CPRIT Single Cell Genomics Center Grant (RP180684). A Photographs of representative mice of control antibody and anti-CD5L antibody (H-447 and R-35) treated groups. Google Scholar. 4F). E, F Tube formation; scale bar=500m (E) and cell migration; scale bar=200m (F) of RF24 cells treated with either control antibody alone, control antibody+CD5L protein, or R-35 antibody+CD5L protein. Tumor weights and the number of tumor nodules for each group were compared by using either the Student t-test (for comparisons of two groups) if the distribution was normal or MannWhitney if the distribution was not normal. These cells displayed increased proliferation, tube-formation capacity, and cell migration compared with control cells (Fig. No difference was observed between CD36 endothelial-specific KO mice treated with either R-35 antibody or IgG compared with mice treated with WT-R-35 (Fig. Vascular endothelial growth factor (VEGF) inhibition by the addition of bevacizumab to the chemotherapy regimen of metastatic colorectal cancer leads to an improved outcome. Stoltenburg, R., Schubert, T. & Strehlitz, B. 63/004,149 CD5L binding antibodies and uses for the same priority claimfiled byThe University of Texas Systems. Considering our finding that AVA resistance is mediated, in part, by overexpression of CD5L, we next aimed to develop an antibody to specifically target CD5L. Google Scholar. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. The final reaction mixture contained a respective amount of aptamer sequences and a constant 1nM labeled CD5L or VEGF. B Photographs of representative mice treated with scramble aptamer, scramble aptamer+B20, CD5L aptamer (S76.T)+IgG, and S76.T+B20. Correspondence to Ramucirumab, an anti-VEGFR2 antibody, has shown efficacy in GC, but the benefits are limited, in part due to MET-mediated resistance. For human ovarian cancer specimens, CD5L expression was determined according to standard immunohistochemical procedures as previously described in ref. 2G). M CD5L WT promoter construct activation in RF24 endothelial cells cultured in hypoxic and normoxic conditions. Similarly, treatment of HUVECs with CD5L recombinant protein resulted in increased capillary formation and sprout length. S19AD. 24. Here, we present data implicating CD5L involvement in adaptive resistance to bevacizumab. CD5L was diluted from 2000 nM to 0.06nM in 50mM Tris-HCl pH 7.8, 150mM NaCl, 0.05% Tween 20. Clinical outcome data were available and used to generate KaplanMeier curves (R version 3.4.1) based on stratification of specific histology and CD5L tumor endothelial cell expression scores. Endothelial cells were sorted by FACS using CD31 antibody (Miltenyi Biotec) as a positive selection marker for endothelial cells, according to the manufacture guidance (https://www.miltenyibiotec.com/US-en/applications/all-protocols/isolation-and-cultivation-of-endothelial-cells-from-adult-mouse-brain.html#gref). Cell lysate of RF24 cells was collected after treatment with human recombinant CD5L protein, and activation of AKT signaling was checked by Western blotting by using anti-human CD5L, pAKT, AKT, and PPARG antibodies, followed by appropriate secondary antibodies conjugated with horseradish peroxidase. Compared with controls, endothelial cells with elevated PPARG demonstrated a significant increase in both CD5L mRNA and protein (Fig. The knockdown efficiency of siRNAs against CD36 were previously validated via western blotting using an anti-CD36 antibody (Abcam Inc, REF ab252922). The differences among Avastin, Lucentis and Eylea have to do . An antibody-cytokine fusion molecule combined with chemotherapy induces tumor regression in mice and patients with recurrent glioblastoma by boosting antitumor immunity . CD5L-treated RF24 cells had higher expression of CD36 than did untreated RF24 cells (Fig. Anti-VEGF antibody and mouse sFlt-1/Fc fusion protein (soluble VEGFR1) Both treatments act to reduce circulating VEGF: VEGFA: 3.25 and 32.5 pM, IV, single dose: Both treatments induced proteinuria by 3 h after administration, which resolved after 24 h. Both treatments resulted in . : investigation and formal analysis; C.L. Cells were then reconstituted in serum-free Hanks balanced salt solution (Life Technologies). One source was from CD5L immunized rabbit single B cells. C CD5L promoter constructs activation using RF24 endothelial cells containing PPARG overexpressing plasmid versus EV. are inventors for U.S. patent No. For in vivo injections, cells were first washed with PBS twice, followed by trypsinization and centrifugation at under 220g for 5min at 4C. : formal analysis, visualization, methodology, and writingreview and editing; E.B. 3A). Cells (5103) were seeded in 96-well plates and allowed to adhere overnight at 37C. To enhance the specificity of aptamers for the target protein, the RNA pool was incubated with His-tagged counter-selection protein for 30min (counter-selection step). With that, strategies aimed at targeting CD5L could be of benefit to patients treated with antiangiogenic drugs. : formal analysis and validation; A.C.-R.: formal analysis; Z.K. CD5L - KN2.0, Human gene knockout kit via CRISPR, non-homology mediated construct KN406528 Origene, and PPARG - KN2.0, Human gene knockout kit via CRISPR, non-homology mediated KN401538 Origene) were used to generate the stable knockout of CD5L or PPARG in RF24 endothelial cells. 5B, C). Regardless of the exact mechanism, the phenomenon of tumor endothelial cells adapting to their specific microenvironment is now well accepted. In addition, tumors from 116 patients with serous ovarian cancer diagnosed between January 1, 1985, and March 31, 2004, were evaluated with the use of a tissue microarray (TMA) obtained from Wayne State University. The DNA library was prepared as described in refs. In addition, we found that CD5L and HIF1 protein expression levels were also increased at longer incubation times of CoCl2 (Fig. Treatment 3: RF24 cells were simultaneously treated with both CD5L protein and CD5L aptamer and then directly added to each well. Kenji Ichikawa, Saori Watanabe Miyano, Yasuhiro Funahashi, Tetsuo Mashima, Takeru Wakatsuki, Kensei Yamaguchi, Minsik Park, Joohwan Kim, Young-Myeong Kim, Nature Communications The single-cell data were submitted to GEO under the accession number GSE181955. Bergers, G. & Hanahan, D. Modes of resistance to anti-angiogenic therapy. S1C, D). After dissociation, cells were washed with complete media to stop the reaction, centrifuged, and resuspended in freeze media (90% FBS/ 10% DMSO) and frozen at 80C. S6A). was supported by the T32 CA101642 from the NIH. A human-nave scFv phage display library (1010 diversity) was also used as another source for the selection of monoclonal antibodies against CD5L. Briefly, we first filtered the matrix based on minimum/maximum cut-offs for genes/cell, cells/gene, and optional parameters such as mitochondrial gene UMI count as a percentage of the total and normalized the data. To determine possible mechanisms of CD5L elevation in tumor endothelial cells, we next examined the regulation of CD5L gene transcription. : investigation and formal analysis; H.-J.C.: investigation, formal analysis, validation, investigation, and visualization; M.H. S3) for the transcription factor PPARG. The binding buffer used consisted of 10mM Tris-HCl 7.5, 150mM NaCl, 1mM MgCl2, 0.1% Triton. No. Son, N. H. et al. Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L. All studies were approved and supervised by the MD Anderson Institutional Animal Care and Use Committee (IACUC). E Cell proliferation in RF24 endothelial cells containing CD5L-overexpressing plasmid versus EV. Cell culture was performed at 37C in a 5% CO2 incubator with 95% humidity. Importantly, knockdown of CD36 in RF24 cells decreased pAKT level even with CD5L treatment (Fig. N.Z., Z.A., A.K.S.,Z.K., and H.D. : Consulting (Merck, Kiyatec, AstraZeneca, Onxeo, Iylon, ImmunoGen, GSK), shareholder (BioPath), research support (M-Trap). Next, to further explore the function of PPARG and CD5L in tumor endothelial cells, we injected murine ID8 ovarian cancer cells into the peritoneal cavity of C57BL/6 mice containing an endothelial cellspecific PPARG knockout or into C57BL/6 WT mice (Fig. Western blotting was performed two times as technical replicates; in each repeat, the blotting, including loading control, was performed using the same sample processing controls. 3G). With the use of our initial gene expression dataset generated from AVA-resistant endothelial cells, we performed an ingenuity pathway analysis and found a close correlation with hypoxia signaling proteins (HIF1, EPAS1, and ARNT) (Fig. 24. H&E sections of lung, liver, kidney, and spleen tissue were examined by two in-house pathologists. 267, 275301 (1996). H Concentration of CD5L in media collected from RF24 endothelial cells containing CD5L-overexpressing plasmid versus EV (n=2 biologically independent experiments). We also examined the . Wild type C57BL/6mice were treated either with control IgG or R-35 for two weeks (once a week). It has also been shown to be involved in a variety of cellular processes, including atherosclerosis, infection, and cancer7. F CD5L promoter construct activation using RF24 endothelial cells treated with siPPARG versus siControl. Upon the first SELEX, we identified four sequences (S5, S47, S63, and S72) from the phylogenetic tree that could potentially bind recombinant CD5L (Fig. We selected the S76.T RNA-aptamer, which is a 2-fluoro pyrimidine RNA-aptamer targeting CD5L. Data represented as mean valuesSD, determined by two-tailed Students t-test; n=34 mice for D, E, and G; n=5 mice for B, C, and F. We next performed a survival analysis of PPARG KO mice versus WT mice with concurrent anti-VEGF treatment. 26. PubMed Drug Resist. S8A) and human umbilical venous endothelial cells (HUVECs; Fig. To determine the function of CD5L in tumor angiogenesis, we generated CD5L-overexpressing RF24 endothelial cells (Fig. Endothelial cell CD36 optimizes tissue fatty acid uptake. Before each treatment, S76.T and the control aptamers were subjected to a denaturationrenaturation step using the following protocol: 85C for 5min, cool down on the ice for 2min, and warm up to 37C. The DNA template was in vitro transcribed to RNA. ISSN 2041-1723 (online). We recognize potential limitations, such as the lack of endothelial cells on the single-cell data and a need for additional safety testing prior to the clinical development of CD5L targeted therapy. : investigation and formal analysis; H.D. John, S. et al. : Formal analysis, validation, investigation, visualization, methodology, and writing; K.N. Bevacizumab-resistant cells were derived from the original parental RF24 cell line by continuous exposure to bevacizumab. S18A) and significantly inhibited tube formation and cell migration (Fig. J. Clin. : formal analysis, visualization, and validation; S.U. Ther. Then, the variable genes were identified and the data was scaled as previously described (https://hbctraining.github.io/scRNA-seq_online/lessons/06_SC_SCT_normalization.html). Several targeted therapies have shown efficacy in patients with advanced gastric cancer (GC) and gastroesophageal junction adenocarcinoma (GEJC), including anti-angiogenic agents and immune checkpoint inhibitors. Mice were treated intraperitoneally with either PBS or anti-CD5L antibody (10mg/kg) starting on Day 8 after tumor injection until Day 35. The anti-VEGF activity of the bi-AbCap was further evaluated using in vitro assays. Several anti-angiogenic drugs have been approved for cancer treatment, alone or in combination with other anti-tumoral agents, and anti-angiogenic therapy is essentially an anti-vascular endothelial growth factor (VEGF) or anti-VEGF-receptor (VEGFR) therapy [].The first anti-angiogenic drug, bevacizumab (Avastin), a humanized anti-VEGF-A monoclonal antibody, was approved for . : Conceptualization, resources, and supervision; Z.A. The use of monoclonal antibodies targeting the vascular endothelial growth factor (VEGF) pathway has been a significant addition to cancer therapy. 3F). Experiments were performed in triplicate and repeated at least twice. We also thank the MD Anderson Flow Cytometry and Cellular Imaging Core Facility (funded by NCI Cancer Center Support Grant P30 CA16672) and MD Andersons Research Medical Library (Tamara K. Locke) for editing this manuscript. Provided by the Springer Nature SharedIt content-sharing initiative. Although many patients benefit from such therapies, virtually all patients eventually experience relapse or progression of the disease. Similarly, both tube formation and cell migration of RF24 cells increased significantly after CD5L treatment (Fig. S4). Gene expression profiling of B20-sensitive and -resistant tumor endothelial cells also showed that both PPARG and HIF1 expression levels were higher (the difference was significant for HIF1) in resistant endothelial cells compared with sensitive endothelial cells (Fig. VEGFR runs at a HIGHER molecular weight than VEGF. PubMed 3E), possibly through a positive-feedback mechanism. G, H Cell viability of RF24 cells at increasing concentrations of bevacizumab with the addition of either CD5L protein (G) or siCD5L (H). Although CD5L is secreted primarily by macrophages, it has been shown to have diverse roles in the immune system. 7B). The DNA library consisted of a random internal region of 40 base pairs flanked by two constant regions at the 5 and 3 ends for the amplification reaction. RF24 cells were treated with bevacizumab (1mg/ml, IC50) for 72h. This medium was then removed, and the cells were allowed to recover for 7 days.