Smart glypican-3-targeting peptide-chlorin e6 conjugates for targeted photodynamic therapy of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly aggressive and lethal malignancy with poor prognosis, necessitating the urgent development of effective treatments. Targeted photodynamic therapy (PDT) offers a promising way to selectively eradicate tumor cells without affecting normal cells. Inspired by promising features of peptide-drug conjugates (PDCs) in targeted cancer therapy, herein a novel glypican-3 (GPC3)-targeting PDC-PDT strategy was developed for the precise PDT treatment of HCC. The GPC3-targeting photosensitizer conjugates were developed by attaching GPC3-targeting peptides to chlorin e6. Conjugate 8b demonstrated the ability to penetrate HCC cells via GPC3-mediated entry process, exhibiting remarkable tumor-targeting capacity, superior antitumor efficacy, and minimal toxicity towards normal cells. Notably, conjugate 8b achieved complete tumor elimination upon light illumination in a HepG2 xenograft model without harm to normal tissues. Overall, this innovative GPC3-targeting conjugation strategy demonstrates considerable promise for clinical applications for the treatment of HCC.

A novel complement-fixing IgM antibody targeting GPC1 as a useful immunotherapeutic strategy for the treatment of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a very low survival rate at 5 years. The use of chemotherapeutic agents results in only modest prolongation of survival and is generally associated with the occurrence of toxicity effects. Antibody-based immunotherapy has been proposed for the treatment of PDAC, but its efficacy has so far proved limited. The proteoglycan glypican-1 (GPC1) may be a useful immunotherapeutic target because it is highly expressed on the surface of PDAC cells, whereas it is not expressed or is expressed at very low levels in benign neoplastic lesions, chronic pancreatitis, and normal adult tissues. Here, we developed and characterized a specific mouse IgM antibody (AT101) targeting GPC1. METHODS: We developed a mouse monoclonal antibody of the IgM class directed against an epitope of GPC1 in close proximity to the cell membrane. For this purpose, a 46 amino acid long peptide of the C-terminal region was used to immunize mice by an in-vivo electroporation protocol followed by serum titer and hybridoma formation. RESULTS: The ability of AT101 to bind the GPC1 protein was demonstrated by ELISA, and by flow cytometry and immunofluorescence analysis in the GPC1-expressing "PDAC-like" BXPC3 cell line. In-vivo experiments in the BXPC3 xenograft model showed that AT101 was able to bind GPC1 on the cell surface and accumulate in the BXPC3 tumor masses. Ex-vivo analyses of BXPC3 tumor masses showed that AT101 was able to recruit immunological effectors (complement system components, NK cells, macrophages) to the tumor site and damage PDAC tumor tissue. In-vivo treatment with AT101 reduced tumor growth and prolonged survival of mice with BXPC3 tumor (p < 0.0001). CONCLUSIONS: These results indicate that AT101, an IgM specific for an epitope of GPC1 close to PDAC cell surface, is a promising immunotherapeutic agent for GPC1-expressing PDAC, being able to selectively activate the complement system and recruit effector cells in the tumor microenvironment, thus allowing to reduce tumor mass growth and improve survival in treated mice.

Glutamine synthetase and hepatocellular carcinoma.

Glutamine synthetase (GS) is an enzyme that converts ammonia and glutamate to glutamine using adenosine triphosphate. GS is expressed in the brain, kidney, and liver tissues under normal physiological conditions. GS is involved in abnormal lipid metabolism of the liver by catalyzing de novo synthesis of glutamine, thereby inducing liver inflammation. Metabolic dysfunction-associated steatotic liver diseases (MASLD), such as Metabolic Associated Fatty Liver Disease and Metabolic Associated Steato Hepatitis, are considered risk factors for HCC. GS may also be involved in the development and progression of hepatocellular carcinoma (HCC) through other signaling pathways, including the rapamycin (mTOR) and Wnt/ß-catenin signaling pathways. Furthermore, the correct combination of HSP70, GPC3, and GS can improve the accuracy and precision of HCC diagnosis. However, the prognostic value of GS in different HCC populations remains controversial. The expression of GS affects the sensitivity of HCC cells to radiotherapy and chemotherapy. In addition, immunotherapy has been approved for the treatment of advanced HCC. This article delves into the development and application of GS in HCC, laying a theoretical foundation for the subsequent exploration of GS as a potential target for treating HCC.

The Role of microRNAs in Regulating Cancer Cell Response to Oxaliplatin-Containing Regimens.

Oxaliplatin (cyclohexane-1,2-diamine; oxalate; platinum [2+]) is a third-generation chemotherapeutic drug with anticancer effects. Oxaliplatin has a role in the treatment of several cancers. It is one of the few drugs which can eliminate the neoplastic cells of colorectal cancer. Also, it has an influential role in breast cancer, lung cancer, bladder cancer, prostate cancer, and gastric cancer. Although oxaliplatin has many beneficial effects in cancer treatment, resistance to this drug is in the way to cure neoplastic cells and reduce treatment efficacy. microRNAs are a subtype of small noncoding RNAs with ∼22 nucleotides that exist among species. They have diverse roles in physiological processes, including cellular proliferation and cell death. Moreover, miRNAs have essential roles in resistance to cancer treatment and can strengthen sensitivity to chemotherapeutic drugs and regimens. In colorectal cancer, the co-treatment of oxaliplatin with anti-miR-19a can partially reverse the oxaliplatin resistance through the upregulation of phosphatase and tensin homolog (PTEN). Moreover, by preventing the spread of gastric cancer cells and downregulating glypican-3 (GPC3), MiR-4510 may modify immunosuppressive signals in the tumor microenvironment. Treatment with oxaliplatin may develop into a specialized therapeutic drug for patients with miR-4510 inhibition and glypican-3-expressing gastric cancer. Eventually, miR-122 upregulation or Wnt/ß-catenin signaling suppression boosted the death of HCC cells and made them more sensitive to oxaliplatin. Herein, we have reviewed the role of microRNAs in regulating cancer cells' response to oxaliplatin, with particular attention to gastrointestinal cancers. We also discussed the role of these noncoding RNAs in the pathophysiology of oxaliplatin-induced neuropathic pain.

Preclinical optimization of a GPC2-targeting CAR T-cell therapy for neuroblastoma.

BACKGROUND: Although most patients with newly diagnosed high-risk neuroblastoma (NB) achieve remission after initial therapy, more than 50% experience late relapses caused by minimal residual disease (MRD) and succumb to their cancer. Therapeutic strategies to target MRD may benefit these children. We developed a new chimeric antigen receptor (CAR) targeting glypican-2 (GPC2) and conducted iterative preclinical engineering of the CAR structure to maximize its anti-tumor efficacy before clinical translation. METHODS: We evaluated different GPC2-CAR constructs by measuring the CAR activity in vitro. NOD-SCID mice engrafted orthotopically with human NB cell lines or patient-derived xenografts and treated with human CAR T cells served as in vivo models. Mechanistic studies were performed using single-cell RNA-sequencing. RESULTS: Applying stringent in vitro assays and orthotopic in vivo NB models, we demonstrated that our single-chain variable fragment, CT3, integrated into a CAR vector with a CD28 hinge, CD28 transmembrane, and 4-1BB co-stimulatory domain (CT3.28H.BBζ) elicits the best preclinical anti-NB activity compared with other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8+ effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as GNLY, GZMB, ZNF683, and HMGN2. Finally, we also showed that the CT3.28H.BBζ CAR we developed was more potent than a recently clinically tested GD2-targeted CAR to control NB growth in vivo. CONCLUSION: Given the robust preclinical activity of CT3.28H.BBζ, these results form a promising basis for further clinical testing in children with NB.

Enhancing anti-tumor efficacy and immune memory by combining 3p-GPC-3 siRNA treatment with PD-1 blockade in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is associated with a high mortality rate and presents a major challenge for human health. Activation of multiple oncogenes has been reported to be strongly associated with the progression of HCC. Moreover, the immunosuppressive tumor microenvironment (TME) and the host immune system are also implicated in the development of malignant HCC tumors. Glypican-3 (GPC-3), a proteoglycan involved in the regulation of cell proliferation and apoptosis, is aberrantly expressed in HCC. We synthesized a short 5'-triphosphate (3p) RNA targeting GPC-3, 3p-GPC-3 siRNA, and found that it effectively inhibited subcutaneous HCC growth by raising type I IFN levels in tumor cells and serum and promoting tumor cell apoptosis. Moreover, 3p-GPC-3 siRNA was able to enhance the activation of CD4+ T cells, CD8+ T cells, and natural killer (NK) cells while reducing the proportion of regulatory T cells (Tregs) in the TME. Most intriguingly, a blocking anti-PD-1 antibody improved the anti-tumor effect of 3p-GPC-3 siRNA, predominantly by activating the immune response, reversing immune exhaustion, and improving immune memory. Our study suggests that the combination of 3p-GPC-3 siRNA administration and PD-1 blockade may represent a promising therapeutic strategy for HCC.

pH-activated nanoplatform for visualized photodynamic and ferroptosis synergistic therapy of tumors.

To overcome drug resistance and improve precision theranostics for hepatocellular carcinoma (HCC), a nanoplatform with an "off/on" function for multimodality imaging (near-infrared-II (NIR-II) fluorescence imaging, magnetic resonance imaging (MRI), and photoacoustic imaging) and synergistic therapy (photodynamic therapy and ferroptosis) activated by an acidic pH in the tumor microenvironment is proposed. Although many photosensitizers with photodynamic effects have been reported, very few of them have outstanding photodynamic effect and high stability with response to endogenous stimuli capable of NIR-II imaging. Herein, a new amphiphilic photosensitizer SR780 derived from croconaine dye, was developed with satisfactory photodynamic effects and pH-responsive NIR-II imaging. Interestingly, it was deactivated by coordination with Fe3+ (SR780@Fe) and activated during their release under mild acidic condition. Ferroptosis can generate hydroxyl free radical and lipid peroxide, which aggravate the oxidative stress of tumor cells and mediate their death while depleting glutathione (GSH) to enhance photodynamic effect. In situ pH-activatable theranostic nanoplatform, SR780@Fe-PAE-GP, was thus developed by loading SR780@Fe with pH-responsive polymers, modified by a glypican-3 (GPC-3) receptor-targeting peptide. The synergistic antitumor effects were confirmed both in vitro and in vivo, and the tumor inhibition rate of the SR780@Fe-PAE-GP + L treatment group reached 98%.

A New Combination: Anti Glypican-3 scFv and Diphtheria Toxin with the Best Flexible Linker.

Along with all cancer treatments, including chemotherapy, radiotherapy, and surgery, targeting therapy is a new treatment manner. Immunotoxins are new recombinant structures that kill cancer cells by targeting specific antigens. Immunotoxins are composed of two parts: toxin moiety, which disrupts protein synthesis process, and antigen binding moiety that bind to antigens on the surface of cancer cells. Glypican 3 (GPC3) is an oncofetal antigen on the surface of Hepatocellular carcinoma (HCC) cells. In this study, truncated Diphtheria toxin (DT389) was fused to humanized scFv YP7 by one, two and three repeats of GGGGS linkers (DT389-(GGGGS)1-3YP7). In-silico and experimental investigation were performed to find out how many repeats of linker between toxin and scFv moieties are sufficient. Results of in-silico investigations revealed that the difference in the number of linkers does not have a significant effect on the main structures of the immunotoxin; however, the three-dimensional structure of two repeats of linker had a more appropriate structure compared to others with one and three linker replications. In addition, with enhancing the number of linkers, the probability of protein solubility has increased. Generally, the bioinformatics results of DT389-(GGGGS)2-YP7 structure showed that expression and folding is suitable; and YP7 scFv has appropriate orientation to bind GPC3. The experimental investigations indicated that the fusion protein was expressed as near to 50% soluble. Due to the high binding affinity of YP7 scFv and the proven potency of diphtheria in inhibiting protein synthesis, the proposed DT389-(GGGGS)2-YP7 immunotoxin is expected to function well in inhibiting HCC.

Nanoparticles Loaded with GSK1059615 Combined with Sorafenib Inhibited Programmed Cell Death 1 Ligand 1 Expression by Negatively Regulating the PI3K/Akt/NF-κB Pathway, Thereby Reversing the Drug Resistance of Hepatocellular Carcinoma to Sorafenib.

Activation of the cellular signaling pathways can induce sorafenib-resistant hepatocellular carcinoma (HCCR). In this work, the PI3K/mTOR inhibitor GSK1059615 inhibited the proliferation and invasion of HCCR cells. PLGA-PEG-mal diblock copolymer was used to load GSK1059615 and sorafenib, and the vector was further modified with GPC3 antibody (hGC33) to obtain hGC33-modified GSK1059615 and sorafenib-loaded nanoparticles (Ab-G/S-NP). Ab-G/S-NP regulated the activation of cellular signaling pathways in HCCR cells by inhibiting the expression and activation of NF-κB and downregulating the level of programmed cell death 1 ligand 1(PD-L1) to reverse drug resistance of HCCR cells to sorafenib. These findings deserve further study in the combined treatment of HCCR cells with GSK1059615 in vivo to develop a more effective treatment of sorafenib-resistant cancers.

The efficacy and adverse effects of nivolumab and lenvatinib in the treatment of advanced hepatocellular carcinoma.

This study intends to investigate nivolumab's efficacy and adverse effects in combination with lenvatinib in treating advanced hepatocellular carcinoma (HCC). For this purpose, ninety-two patients with unresectable advanced HCC admitted were enrolled and were divided into the control group (N=46) and the observation group (N=46) according to the random number table. The control group was treated with lenvatinib while the observation group was treated with nivolumab combined with lenvatinib. The efficacy, adverse effects, liver function, completion rate, interruption and discontinuation of treatment, drug reduction, serum tumor markers, and immune function were compared between the two groups. Also, changes in the expression of some genes that regulate the cell cycle (P53, RB1, Cyclin-D1, c-fos, and N-ras) were investigated in the development of this cancer. According to the results, ORR and DCR  (45.65%, 78.26%) in the observation group were higher than those  (23.91%, 54.35%) in the control group (P<0.05); The incidence of adverse reactions in the observation group was slightly higher than that of the control group, but the difference was not significant (P>0.05); The rate of completion, interruption, discontinuation of treatment and drug reduction did not differ significantly between two groups (P>0.05); After treatment, the serum ALT, AST, TBIL, and GGT levels decreased and were lower in observation group than in control group (P<0.05); The serum tumor markers AFP, ENO1, GPC3, CEA levels decreased in both groups after treatment, and were lower in the observation group than in control group (P<0.05);  CD3, CD4, CD8, and NK levels were improved in the observation group and worsened in the control group, and CD3, CD4, and NK levels were higher in the observation group and lower in the control group after treatment (P<0.05). All in all, nivolumab combined with lenvatinib for advanced hepatocellular carcinoma can improve tumor control, reduce tumor load, and improve liver function and immune function. Common adverse reactions include fatigue, loss of appetite, elevated blood pressure, hand-foot skin reaction, diarrhea, and rash, which should be controlled during treatment.

Glypican-1 Is a Novel Target for Stroma and Tumor Cell Dual-Targeting Antibody-Drug Conjugates in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a stroma-rich cancer. Extracellular matrix proteins produced by cancer-associated fibroblasts (CAFs) found in tumor stroma that impedes effective delivery of chemotherapeutic agents results in poor response in patients with PDAC. Previously, our group reported that glypican-1 (GPC1) was overexpressed in human PDAC and negatively correlated with patient survival. Immunohistochemical analysis of 25 patients with PDAC tumor specimens revealed elevated expression of GPC1 in stromal cells and pancreatic cancer cells in 80% of patients. Interestingly, GPC1 was expressed on CAFs in PDAC. We generated a GPC1 antibody-drug conjugate conjugated with monomethyl auristatin E [GPC1-ADC(MMAE)] and evaluated its preclinical antitumor activity by targeting GPC1-positive CAF and cancer cells in PDAC. GPC1-ADC(MMAE) inhibited the growth of GPC1-positive PDAC cell lines in vitro Furthermore, GPC1-ADC(MMAE) showed a potent antitumor effect in the PDAC patient-derived tumor xenograft (PDX) model against GPC1-positive CAF and heterogeneous GPC1-expressing cancer cells. Notably, GPC1-ADC(MMAE) showed robust preclinical efficacy against GPC1 in a stroma-positive/cancer-negative PDAC PDX model. GPC1-ADC(MMAE) was delivered and internalized to CAFs. Although apoptosis was not observed in CAFs, the released MMAE from CAFs via MDR-1 induced apoptosis of cancer cells neighboring CAFs and efficiently inhibited PDAC tumor growth. GPC1-ADC(MMAE) exhibited potent and unique antitumor activity in GPC1-positive PDAC PDX models, which suggests that GPC1 is a novel therapeutic target in PDAC and other stromal GPC1-positive solid tumors. These findings show that targeting GPC1 on CAF using GPC1-ADC(MMAE) is a useful approach in case of stroma-rich tumors such as PDAC.

A novel targeted GPC3/CD3 bispecific antibody for the treatment hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer but has shown limited success to date in the treatment of advanced stage. Recruitment of T cells for cancer treatment is a rapidly growing strategy in immunotherapy such as chimeric antigen receptor T cells and bispecific antibodies. However, unwanted aggregations, structural instability or short serum half-life are major challenges of bispecific antibodies. Here, we developed a new format of T cell-redirecting antibody that is bispecific for membrane proteoglycans GPC3 of HCC and the T-cell-specific antigen CD3, which demonstrated to be favorable stability and productivity. Cross-linking of T cells with GPC3 positive tumor cells by the anti-GPC3/CD3 bispecific antibody-mediated potent GPC3-dependent and concentration-dependent cytotoxicity in vitro. Administration of the bispecific antibody with different concentrations in murine xenograft models of human HCC significantly inhibited tumor growth. In addition, no effects on tumor growth were observed in the absence of human effector cells or the bispecific antibody. Taken together, the anti-GPC3/CD3 bispecific antibody might be a potential therapeutic treatment for HCC.

Titania nanotube-based protein delivery system to inhibit cranial bone regeneration in Crouzon model of craniosynostosis.

BACKGROUND: Craniosynostosis is a developmental disorder characterized by the premature fusion of skull sutures, necessitating repetitive, high-risk neurosurgical interventions throughout infancy. This study used protein-releasing Titania nanotubular implant (TNT/Ti) loaded with glypican 3 (GPC3) in the cranial critical-sized defects (CSDs) in Crouzon murine model (Fgfr2c342y/+ knock-in mutation) to address a key challenge of delaying post-operative bone regeneration in craniosynostosis. MATERIALS AND METHODS: A 3 mm wide circular CSD was created in two murine models of Crouzon syndrome: (i) surgical control (CSDs without TNT/Ti or any protein, n=6) and (ii) experimental groups with TNT/Ti loaded with GPC3, further subdivided into the presence or absence of chitosan coating (on nanotubes) (n=12 in each group). The bone volume percentage in CSDs was assessed 90 days post-implantation using micro-computed tomography (micro-CT) and histological analysis. RESULTS: Nano-implants retrieved after 90 days post-operatively depicted well-adhered, hexagonally arranged, and densely packed nanotubes with average diameter of 120±10 nm. The nanotubular architecture was generally well-preserved. Compared with the control bone volume percentage data (without GPC3), GPC3-loaded TNT/Ti without chitosan coating displayed a significantly lower volume percent in cranial CSDs (P<0.001). Histological assessment showed relatively less bone regeneration (healing) in GPC3-loaded CSDs than control CSDs. CONCLUSION: The finding of inhibition of cranial bone regeneration by GPC3-loaded TNT/Ti in vivo is an important advance in the novel field of minimally-invasive craniosynostosis therapy and holds the prospect of altering the whole paradigm of treatment for affected children. Future animal studies on a larger sample are indicated to refine the dosage and duration of drug delivery across different ages and both sexes with the view to undertake human clinical trials.

Redirecting T Cells to Glypican-3 with 4-1BB Zeta Chimeric Antigen Receptors Results in Th1 Polarization and Potent Antitumor Activity.

T cells engineered to express CD19-specific chimeric antigen receptors (CARs) have shown breakthrough clinical successes in patients with B-cell lymphoid malignancies. However, similar therapeutic efficacy of CAR T cells in solid tumors is yet to be achieved. In this study we systematically evaluated a series of CAR constructs targeting glypican-3 (GPC3), which is selectively expressed on several solid tumors. We compared GPC3-specific CARs that encoded CD3ζ (Gz) alone or with costimulatory domains derived from CD28 (G28z), 4-1BB (GBBz), or CD28 and 4-1BB (G28BBz). All GPC3-CARs rendered T cells highly cytotoxic to GPC3-positive hepatocellular carcinoma, hepatoblastoma, and malignant rhabdoid tumor cell lines in vitro. GBBz induced the preferential production of Th1 cytokines (interferon γ/granulocyte macrophage colony-stimulating factor) while G28z preferentially induced Th2 cytokines (interleukin-4/interleukin-10). Inclusion of 4-1BB in G28BBz could only partially ameliorate the Th2-polarizing effect of CD28. 4-1BB induced superior expansion of CAR T cells in vitro and in vivo. T cells expressing GPC3-CARs incorporating CD28, 4-1BB, or both induced sustained tumor regressions in two xenogeneic tumor models. Thus, GBBz CAR endows T cells with superior proliferative potential, potent antitumor activity, and a Th1-biased cytokine profile, justifying further clinical development of GBBz CAR for immunotherapy of GPC3-positive solid tumors.

Prediction and preliminary screening of HLA-A*0201-restricted epitope peptides of human GPC3.

In response to the limited therapeutic option for hepatocellular carcinoma (HCC), immunotherapy as a promising approach points out a new direction to the cure of tumours through specific recognition and elimination of tumour cells by the immunity-enhanced autologous immunocytes of patients. Few effective tumour antigens, however, are alternative in addition to alpha fetoprotein or tumour cell lysates. Recent studies have demonstrated that glypican-3 (GPC3) is not only a promising diagnostic marker, but also ideal therapeutic target to HCC. In this study, potential HLA-A*0201 GPC3 peptides were screened with three epitope prediction software, the binding affinity of 13 predicted epitopes with high scores was determined by T2 cells binding assay and four optimal epitopes were identified. This is the first study in which the optimal HLA-A*0201 GPC3 epitopes were screened from a large number of candidates predicted by three software. The optimized HLA-A*0201 GPC3 peptides will provide new epitope candidates for HCC immunotherapy.

Programmed death-1 blockade enhances the antitumor effects of peptide vaccine-induced peptide-specific cytotoxic T lymphocytes.

Novel treatment modalities are required urgently in patients with hepatocellular carcinoma (HCC). A vaccine that induces cytotoxic T lymphocytes (CTLs) is an ideal strategy for cancer, and glypican-3 (GPC3) is a potential option for HCC. Blocking the programmed death-1 (PD-1)/PD-L1 pathway is a rational strategy to overcome tumor escape and tolerance toward CTLs. In the present study, we investigated whether anti-PD-1 blocking antibodies (αPD-1 Ab) enhanced the number of vaccine-induced peptide-specific CTLs in peripheral blood mononuclear cells (PBMCs) following the administration of GPC3 peptide vaccine to both patients and in a mouse model. The inhibitory receptor PD-1 was highly expressed in ex vivo GPC3-specific CTLs isolated from the PBMCs of vaccinated HCC patients. In vitro, interferon-γ induced PD-L1 expression in liver cancer cell lines. In addition, PD-1 blockade increased the number of GPC3-specific CTLs, which degranulate against liver cancer cell lines. In vivo experiments using tumor-bearing mouse models showed that the combination therapy of peptide vaccine and αPD-1 Ab suppressed tumor growth synergistically. PD-1 blockade increased the number of peptide-specific tumor-infiltrating T cells (TILs) and decreased the expression of inhibitory receptors on TILs. This study demonstrated that PD-1/PD-L1 blockade augmented the antitumor effects of a peptide vaccine by increasing the immune response of vaccine-induced CTLs, and provided a foundation for the clinical development of a combination therapy using a GPC3 peptide vaccine and αPD-1 Ab.

Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats.

Physical and chemical constraints imposed by the periinfarct glial scar may contribute to the limited clinical improvement often observed after ischemic brain injury. To investigate the role of some of these mediators in outcome from cerebral ischemia, we treated rats with the growth-inhibitory chondroitin sulfate proteoglycan neurocan, the growth-stimulating heparan sulfate proteoglycan glypican, or the chondroitin sulfate proteoglycan-degrading enzyme chondroitinase ABC. Neurocan, glypican, or chondroitinase ABC was infused directly into the infarct cavity for 7 d, beginning 7 d after middle cerebral artery occlusion. Glypican and chondroitinase ABC reduced glial fibrillary acidic protein immunoreactivity and increased microtubule-associated protein-2 immunoreactivity in the periinfarct region, and glypican- and chondroitinase ABC-treated rats showed behavioral improvement compared with neurocan- or saline-treated rats. Glypican and chondroitinase ABC also increased neurite extension in cortical neuron cultures. Glypican increased fibroblast growth factor-2 expression and chondroitinase ABC increased brain-derived neurotrophic factor expression in these cultures, whereas no such effects were seen following neurocan treatment. Thus, treatment with glypican or enzymatic disruption of neurocan with chondroitinase ABC improves gross anatomical, histological, and functional outcome in the chronic phase of experimental stroke in rats. Changes in growth factor expression and neuritogenesis may help to mediate these effects.