IT-141, a Polymer Micelle Encapsulating SN-38, Induces Tumor Regression in Multiple Colorectal Cancer Models

Polymer micelles are promising drug delivery vehicles for the delivery of anticancer agents to tumors. Often, anticancer drugs display potent cytotoxic effects towards cancer cells but are too hydrophobic to be administered in the clinic as a free drug. To address this problem, a polymer micelle was designed using a triblock copolymer (ITP-101) that enables hydrophobic drugs to be encapsulated. An SN-38 encapsulated micelle, IT-141, was prepared that exhibited potent in vitro cytotoxicity against a wide array of cancer cell lines. In a mouse model, pharmacokinetic analysis revealed that IT-141 had a much longer circulation time, plasma exposure, and tumor exposure compared to irinotecan. IT-141 was also superior to irinotecan in terms of antitumor activity, exhibiting greater tumor inhibition in HT-29 and HCT116 colorectal cancer xenograft models at half the dose of irinotecan. The antitumor effect of IT-141 was dose-dependent and caused complete growth inhibition and tumor regression at well-tolerated doses. Varying the specific concentration of SN-38 within the IT-141 micelle had no detectible effect on this antitumor activity, indicating no differences in activity between different IT-141 formulations. In summary, IT-141 is a potent micelle-based chemotherapy that holds promise for the treatment of colorectal cancer

A Versatile Polymer Micelle Drug Delivery System for Encapsulation and In Vivo Stabilization of Hydrophobic Anticancer Drugs

Chemotherapeutic drugs are widely used for the treatment of cancer; however, use of these drugs is often associated with patient toxicity and poor tumor delivery. Micellar drug carriers offer a promising approach for formulating and achieving improved delivery of hydrophobic chemotherapeutic drugs; however, conventional micelles do not have long-term stability in complex biological environments such as plasma. To address this problem, a novel triblock copolymer has been developed to encapsulate several different hydrophobic drugs into stable polymer micelles. These micelles have been engineered to be stable at low concentrations even in complex biological fluids, and to release cargo in response to low pH environments, such as in the tumor microenvironment or in tumor cell endosomes. The particle sizes of drugs encapsulated ranged between 30–80 nm, with no relationship to the hydrophobicity of the drug. Stabilization of the micelles below the critical micelle concentration was demonstrated using a pH-reversible crosslinking mechanism, with proof-of-concept demonstrated in both in vitro and in vivo models. Described herein is polymer micelle drug delivery system that enables encapsulation and stabilization of a wide variety of chemotherapeutic drugs in a single platform

Proteolytic cleavage of E-cadherin and beta-catenin by calpain in prostate and mammary tumor cells.

The adherens junction, which includes E-cadherin and beta-catenin, maintains inter-cellular adhesion between adjacent epithelial cells and also mediates intracellular signals. Regulation of components of the adherens junction is important in cell survival and cell death. This dissertation reports a novel mechanism of E-cadherin inactivation through calpain-mediated cleavage of E-cadherin and generation of a stable 100-kDa fragment (E-cad100 ). This fragment was generated in prostate and mammary tumor cells through PKC activation or calcium influx and lacked the beta-catenin binding domain. Mutagenesis revealed a six amino-acid sequence in the E-cadherin cytoplasmic domain that was required for cleavage. E-cad100 fused to GFP did not associate or co-localize with endogenous beta-catenin in LNCaP cells. Because PKC activation induced aggregation-dependent survival of LNCaP cells that was dependent on functional E-cadherin, we hypothesized that overexpression of beta-catenin-binding deficient E-cad100 might play an inhibitory role. Overexpression of E-cad100 downregulated endogenous E-cadherin and potentiated PKC-induced cell death. These results demonstrated that calpain cleaves E-cadherin during PKC-induced apoptosis and overexpression of E-cad100 potentiates cell death. Proteolysis of E-cadherin and elevated m-calpain expression were observed in localized and metastatic prostate cancer specimens compared to benign prostate tissue. Further analysis also detected a 75-kDa beta-catenin fragment (beta-cat 75), which lacked the N-terminal regulatory domain, in metastatic prostate cancer tissue as well as several prostate and breast cancer cell lines. Calcium influx induced the calpain-dependent generation of beta-cat 75 that accumulated in nuclear and cytosolic compartments. Overexpression of a similar N-terminally truncated beta-catenin mutant activated TCF-dependent transcription. It is proposed that the loss of the N-terminal regulatory domain imposes greater stability and oncogenicity on beta-cat75, based on previous studies which demonstrated that similar N-terminal truncation mutants of beta-catenin are highly stable and tumorigenic. These results elucidate a role for calpain in regulation of E-cadherin and beta-catenin in both cell death and cell survival pathways. This dissertation describes a novel, calpain-mediated mechanism for the inactivation of E-cadherin and activation of beta-catenin in prostate and mammary tumor cells.Ph.D.Biological SciencesCellular biologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124544/2/3150075.pd

Identification and validation of dichotomous immune subtypes based on intratumoral immune cells infiltration in clear cell renal cell carcinoma patients

BackgroundIncreasing evidence has elucidated the clinical significance of tumor infiltrating immune cells in predicting outcomes and therapeutic efficacy. In this study, we comprehensively analyze the tumor microenvironment (TME) immune cell infiltrations in clear cell renal cell carcinoma (ccRCC) and correlated the infiltration patterns with anti-tumor immunity and clinical outcomes.MethodsWe analyzed immune cell infiltrations in four independent cohorts, including the KIRC cohort of 533 patients, the Zhongshan ccRCC cohorts of 259 patients, the Zhongshan fresh tumor sample cohorts of 20 patients and the Zhongshan metastatic ccRCC cohorts of 87 patients. Intrinsic patterns of immune cell infiltrations were evaluated for associations with clinicopathological characteristics, underlying biological pathways, genetic changes, oncological outcomes and treatment responses.ResultsUnsupervised clustering of tumor infiltrating immune cells identified two microenvironment subtypes, TMEcluster-A and TMEcluster-B. Gene markers and biological pathways referring to immune evasion were upregulated in TMEcluster-B. TMEcluster-B associated with poor overall survival (p<0.001; HR 2.629) and recurrence free survival (p=0.012; HR 1.870) in ccRCC validation cohort. TMEcluster-B cases had worse treatment response (p=0.009), overall survival (p<0.001; HR 2.223) and progression free survival (p=0.015; HR 2.7762) in metastatic ccRCC cohort. The predictive accuracy of International Metastatic Database Consortium risk score was improved after incorporation of TME clusters.ConclusionsTMEcluster-A featured increased mast cells infiltration, prolonged survival and better treatment response. TMEcluster-B was a heavily infiltrated but immunosuppressed phenotype enriched for macrophages, CD4+ T cells, Tregs, CD8+ T cells and B cells. TMEcluster-B predicted dismal survival and worse treatment response in clear cell renal cell carcinoma patients

Characterization of human cancer xenografts in humanized mice

BackgroundPreclinical evaluation of drugs targeting the human immune system has posed challenges for oncology researchers. Since the commercial introduction of humanized mice, antitumor efficacy and pharmacodynamic studies can now be performed with human cancer cells within mice bearing components of a human immune system. However, development and characterization of these models is necessary to understand which model may be best suited for different agents.MethodsWe characterized A375, A549, Caki-1, H1299, H1975, HCC827, HCT116, KU-19–19, MDA-MB-231, and RKO human cancer cell xenografts in CD34+ humanized non-obese diabetic-scid gamma mice for tumor growth rate, immune cell profiling, programmed death ligand 1 (PD-L1) expression and response to anti-PD-L1 therapy. Immune cell profiling was performed using flow cytometry and immunohistochemistry. Antitumor response of humanized xenograft models to PD-L1 therapy was performed using atezolizumab.ResultsWe found that CD4+ and CD8+ T-cell composition in both the spleen and tumor varied among models, with A375, Caki-1, MDA-MB-231, and HCC827 containing higher intratumoral frequencies of CD4+ and CD8+ T cells of CD45+ cells compared with other models. We demonstrate that levels of immune cell infiltrate within each model are strongly influenced by the tumor and not the stem cell donor. Many of the tumor models showed an abundance of myeloid cells, B cells and dendritic cells. RKO and MDA-MB-231 tumors contained the highest expression of PD-L1+ tumor cells. The antitumor response of the models to atezolizumab was positively associated with the level of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs).ConclusionsThese data demonstrate that there are tumor-intrinsic factors that influence the immune cell repertoire within tumors and spleen, and that TIL frequencies are a key factor in determining response to anti-PD-L1 in tumor xenografts in humanized mice. These data may also aid in the selection of tumor models to test antitumor activity of novel immuno-oncology or tumor-directed agents