Mouse Tumor Lines Research Articles

Establishment and immune phenotyping of patient-derived glioblastoma models in humanized mice.

Glioblastoma (GBM) is the most aggressive and common type of malignant brain tumor diagnosed in adults. Preclinical immunocompetent mouse tumor models generated using mouse tumor cells play a pivotal role in testing the therapeutic efficacy of emerging immune-based therapies for GBMs. However, the clinical translatability of such studies is limited as mouse tumor lines do not fully recapitulate GBMs seen in inpatient settings. In this study, we generated three distinct, imageable human-GBM (hGBM) models in humanized mice using patient-derived GBM cells that cover phenotypic and genetic GBM heterogeneity in primary (invasive and nodular) and recurrent tumors. We developed a pipeline to first enrich the tumor-initiating stem-like cells and then successfully established robust patient-derived GBM tumor engraftment and growth in bone marrow-liver-thymus (BLT) humanized mice. Multiplex immunofluorescence of GBM tumor sections revealed distinct phenotypic features of the patient GBM tumors, with myeloid cells dominating the immune landscape. Utilizing flow cytometry and correlative immunofluorescence, we profiled the immune microenvironment within the established human GBM tumors in the BLT mouse models and showed tumor infiltration of variable human immune cells, creating a unique immune landscape compared with lymphoid organs. These findings contribute substantially to our understanding of GBM biology within the context of the human immune system in humanized mice and lay the groundwork for further translational studies aimed at advancing therapeutic strategies for GBM.

Pegylation of phenothiazine – A synthetic route towards potent anticancer drugs

IntroductionCancer is a big challenge of the 21 century, whose defeat requires efficient antitumor drugs. ObjectivesThe paper aims to investigate the synergistic effect of two structural building blocks, phenothiazine and poly(ethylene glycol), towards efficient antitumor drugs. MethodsTwo PEGylated phenothiazine derivatives were synthetized by attaching poly(ethylene glycol) of 550 Da to the nitrogen atom of phenothiazine by ether or ester linkage. Their antitumor activity has been investigated on five human tumour lines and a mouse tumor line as well, by determination of IC50. The in vivo toxicity was determined by measuring the LD50 in BALB/c mice by the sequential method and the in vivo antitumor potential was measured by the tumours growth test. The antitumor mechanism was investigated by complexation studies of zinc and magnesium ions characteristic to the farnesyltransferase enzyme, by studies of self-aggregation in the cells proximity and by investigation of the antitumor properties of the acid species resulted by enzymatic cleavage of the PEGylated derivatives. ResultsThe two compounds showed antitumor activity, with IC50 against mouse colon carcinoma cell line comparable with that of the traditional antitumor drugs 5-Fluorouracil and doxorubicin. The phenothiazine PEGylation resulted in a significant toxicity diminishing, the LD50 in BALB/c mice increasing from 952.38 up to 1450 mg/kg, in phenothiazine equivalents. Both compounds inflicted a 92% inhibition of the tumour growth for doses much smaller than LD50. The investigation of the possible tumour inhibition mechanism suggested the nanoaggregate formation and the cleavage of ester bonds as key factors for the inhibition of cancer cell proliferation and biocompatibility improvement. ConclusionPhenothiazine and PEG building blocks have a synergetic effect working for both tumour growth inhibition and biocompatibility improvement. All these findings recommend the PEGylated phenothiazine derivatives as a valuable workbench for a next generation of antitumor drugs.

Keap1 mutation renders lung adenocarcinomas dependent on Slc33a1.

Approximately 20-30% of human lung adenocarcinomas (LUAD) harbor loss-of-function (LOF) mutations in Kelch-like ECH Associated-Protein 1 (KEAP1), which lead to hyperactivation of the nuclear factor, erythroid 2-like 2 (NRF2) antioxidant pathway and correlate with poor prognosis1-3. We previously showed that Keap1 mutation accelerates KRAS-driven LUAD and produces a marked dependency on glutaminolysis4. To extend the investigation of genetic dependencies in the context of Keap1 mutation, we performed a druggable genome CRISPR-Cas9 screen in Keap1-mutant cells. This analysis uncovered a profound Keap1 mutant-specific dependency on solute carrier family 33 member 1 (Slc33a1), an endomembrane-associated protein with roles in autophagy regulation5, as well as a series of functionally-related genes implicated in the unfolded protein response. Targeted genetic and biochemical experiments using mouse and human Keap1-mutant tumor lines, as well as preclinical genetically-engineered mouse models (GEMMs) of LUAD, validate Slc33a1 as a robust Keap1-mutant-specific dependency. Furthermore, unbiased genome-wide CRISPR screening identified additional genes related to Slc33a1 dependency. Overall, our study provides a strong rationale for stratification of patients harboring KEAP1-mutant or NRF2-hyperactivated tumors as likely responders to targeted SLC33A1 inhibition and underscores the value of integrating functional genetic approaches with GEMMs to identify and validate genotype-specific therapeutic targets.

03:18 PM Abstract No. 181 Utility and safety of large core coaxial needle biopsy for biorepository tissue acquisition during MWA of HCC: initial experience in an US Hispanic population

To report our initial experience with the use of a large core (14 G) coaxial needle biopsy during MWA of HCC for Biorepository Tissue acquisition in an US Hispanic patient population with HCC. Between July 2016 – August 2018, 19 US Hispanic patients (3 F, M 16, ages 50-74 mean 65) with HCC where included in this prospective, IRB-approved study. Tumor size ranged from 1.8 cm to 5.2 cm, mean 2.9 cm. A coaxial 13.5 G needle (Achieve) was advanced in the periphery of the tumor under US and/or CT guidance. A 14 G cutting needle was used to obtain two 2 cm cores. Two passes were made in each patient. One pass was cut in two pieces and a 1 cm fragment was sent to pathology for histological examination. The other portions (75%) of tissue were sent to the Biorepository for establishing liver cancer cell lines, tumor lines in mice, and long-term storage. After the biopsy a 15 G Solero MWA probe (Angiodynamics) was advanced through the coaxial needle and the tumors were ablated in a standard fashion. Tract ablation was performed by pulling the coaxial and the ablation needle together. Patients were followed- up by serial imaging and clinics visits Biopsy and ablation was possible in all patients. Histological analysis confirmed HCC in 13 patients (grade 1 (n=5), grade 2 (n=5), grade 3 (n= 3)) . Atypical nodules were seen in 3 patients and 3 biopsies were negative for cancer. One patient developed a severe hypertensive crisis due to accidental thermal injury of the adrenal gland. An arterioportal fistula without active bleeding was noted in the CT scan after the procedure in another patient and it was treated with embolization. During a mean follow-up of 10 months (range 0.5-24) there was no evidence of tumor seeding by serial imaging in any patient. Three patients died. Causes of death were renal failure, pneumonia and complications of liver transplant 2,10 and 20 months respectively after the ablation. Sixteen patients are alive with various tumor responses to the ablations. Large core coaxial needle biopsy during MWA of HCC is a safe and effective method to obtain tissue for histological analyses and biorepository studies.

Mouse ER+/PIK3CAH1047R breast cancers caused by exogenous estrogen are heterogeneously dependent on estrogen and undergo BIM-dependent apoptosis with BH3 and PI3K agents.

Estrogen dependence is major driver of ER + breast cancer, which is associated with PI3K mutation. PI3K inhibition (PI3Ki) can restore dependence on ER signaling for some hormone therapy-resistant ER + breast cancers, but is ineffective in others. Here we show that short-term supplementation with estrogen strongly enhanced Pik3caH1047R-induced mammary tumorigenesis in mice that resulted exclusively in ER + tumors, demonstrating the cooperation of the hormone and the oncogene in tumor development. Similar to human ER + breast cancers that are endocrine-dependent or endocrine-independent at diagnosis, tumor lines from this model retained ER expression but were sensitive or resistant to hormonal therapies. PI3Ki did not induce cell death but did cause upregulation of the pro-apoptotic gene BIM. BH3 mimetics or PI3Ki were unable to restore hormone sensitivity in several resistant mouse and human tumor lines. Importantly however, combination of PI3Ki and BH3 mimetics had a profound, BIM-dependent cytotoxic effect in PIK3CA-mutant cancer cells while sparing normal cells. We propose that addition of BH3 mimetics offers a therapeutic strategy to markedly improve the cytotoxic activity of PI3Ki in hormonal therapy-resistant and ER-independent PIK3CA-mutant breast cancer.

670. A Novel Approach to Expand Antigen Recognition in Chimeric Antigen Receptors

Chimeric antigen receptor (CAR) technology, although very promising, is limited in its application by the availability of target antigens. We propose to expand the pool of potentially targetable cell-surface antigens using the variable lymphocyte receptor (VLR) of the sea lamprey as the antigen recognition region of the CAR. VLRs represent the functional region of the lamprey and hagfish adaptive immune system. They are single chained and variable length, crescent shaped proteins that are produced by assembly of leucine-rich repeat cassettes to form a gene encoding region capable of exceeding 1015 unique variations. Due to their difference in structure compared to Ig based antibodies, VLRs bind antigen in a geometrically dissimilar manner. This unique property of VLRs allows them the ability to bind antigen epitopes that may not typically be bound by scFvs, the result being a potentially expanded repertoire of tumor cell target antigens that may be used in CAR design and application. In our studies, VLRs have been successfully developed from immunized lampreys and target cell specific VLRs have been cloned for several different antigens including VLRs specific for cancer cell lines and purified proteins. This is accomplished using yeast surface display combined with flow sorting and results in monoclonal VLRs specific for the cell line with which the lampreys were immunized. The functionality of the VLR was initially demonstrated in Jurkat cells transduced with a previously generated VLR specific for the B-cell receptor of the mouse tumor line, BCL. CAR protein expression from whole cell lysates of transduced Jurkat cells showed expression of CAR protein. CAR cell surface expression was also confirmed in Jurkat cells using a construct co-expressing GFP preceding a P2A sequence with >90% cells GFP positive. Transduced CAR-Jurkat cells showed upwards of 85% activation in co-culture assays with target cells. In transduced but not co-cultured cells, activation was <5% and activation in naive cells co-cultured with BCL cells was <2%. These results establish the ability of these cells to effectively produce and express VLR-CAR protein. The BCL VLR-CAR construct was also shown to be functional in several different types of cytotoxic effector cells including gamma delta T-cells and NK-92 cells, where target cell killing was increased significantly over non-transduced cells, indicating that target cell specific toxicity is mediated through the VLR-CAR. From these results, showing that VLRs function effectively as the antigen recognition region of the CAR construct we have concluded that VLRs can serve as a unique alternative for directing CAR activity toward specific effector cells.

Abstract C107: Preclinical response of murine syngeneic tumor models to immune checkpoint inhibitor antibodies

Abstract Tumor cell viability and proliferation is regulated by the balance between tumor self-survival and host elimination mechanisms. There are multiple mechanisms whereby tumors successfully evade host immune cell surveillance, permitting continued growth and proliferation. Immunotherapies are aimed at bolstering the host immune system. Delivery of these agents has been shown to actively eliminate disease in a number of patients to date. We evaluated several syngeneic mouse tumor lines for anti-tumor activity with anti-CTLA-4 and/or anti-PD-L1 antibodies, as well as the immunological changes occurring following treatment with these checkpoint inhibitors. The effectiveness of these inhibitors was assessed in the B16.F10 (melanoma), RIF-1 (sarcoma), 4T1-luc (mammary) and CT26 (colon) murine tumor models. Cells or tumor fragments (RIF-1) were implanted SC in the axilla. The anti-CTLA-4 9D9 antibody clone from Bio X Cell (West Lebanon, NH) was employed for responsiveness in each model. All treatments were initiated 3 days post implantation, prior to the appearance of established tumor, at inoculums known to have 100% take rates. The CT26 model was highly responsive with all animals showing tumor regressions and 70% tumor-free survivors at study end. The RIF-1 model was moderately responsive with several mice showing long term progression-free survival and a 56% increase in lifespan vs. the isotype control group. Both the B16.F10 and the 4T1-luc models were non-responsive. Tumor responsiveness to the anti-PD-L1 10F.9G2 antibody clone (Bio X Cell) was also compared in the RIF-1 and CT26 models. In contrast to the anti-CTLA-4 antibody studies, RIF-1 was found to be non-responsive and CT26 was moderately responsive to the anti-PD-L1 antibody. Evaluating immunological endpoints by flow cytometry is critical when assessing the effectiveness of immune checkpoint inhibitors. To this end, the immune profiles of both responsive (CT26) and non-responsive (4T1 and B16.F10) models were examined in the absence or presence of antibody treatments. Initial studies were focused on analysis of T cells (CD4 and CD8) as well as T regulatory cells (Tregs; CD4/CD25/FoxP3) and myeloid derived suppressive cells (MDSC; CD11b/Gr1). The 4T1 model was found to be highly MDSC driven and would be suitable to evaluate agents that target myeloid-derived cell populations. While this model was not very responsive to anti-CTLA-4 treatment, a trend toward decreased MDSCs following treatment was observed with anti-CTLA-4 and not the isotype control antibody. In contrast, the CT26 model was more highly mixed and contained populations of both Tregs and MDSCs. Treatment with anti-CTLA-4 increased the CD8+ T cell population and decreased the Treg cell population with minimal effect on the MSDC population. However, in this same model, anti-PD-L1 treatment increased CD8+ T cells and decreased both Tregs and MDSCs. A more complete mechanistic understanding of how the immune system responds to checkpoint inhibitors permits identification of appropriate models for monotherapy, as well as the rationale for designing drug combination studies. Data enhancing understanding of the above, along with tumor profiling on additional syngeneic models and more flow cytometry multiplexing, is expected from further ongoing research. Citation Format: Maryland Franklin, Matt Thayer, Chris Elders, Dan Saims, Scott Wise. Preclinical response of murine syngeneic tumor models to immune checkpoint inhibitor antibodies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C107.

Abstract 5429: The combination of Apo2L/Trail and a death receptor 5 agonist antibody leads to enhanced antitumor activity in mouse syngeneic tumor models

Abstract Background: Death receptor-5 (DR5, TR2, TRAIL-R2) is a death domain-containing receptor that induces an apoptosis signaling cascade when engaged by Apo2L/TRAIL or a DR5 agonist antibody1. Many tumor cell lines express DR5 and are sensitive to DR5 agonists2. Despite pre-clinical efficacy, several DR5 agonists have failed to advance beyond phase II clinical trials due to poor clinical efficacy. Strong synergystic activity was observed with the combination of Apo2L/TRAIL and AMG655 (DR5 agonist antibody) pre-clinically in vitro and in vivo (manuscript submitted). Although Apo2L/TRAIL and AMG655 were well tolerated in clinical trials when delivered as single agents3, the efficacy and safety profile of combining Apo2L/TRAIL and AMG655 has not been investigated. Objective: To determine a therapeutic index for Apo2L/TRAIL + DR5 agonist antibody combination treatment in mouse syngeneic tumor cell lines and xenograft models. Results: To evaluate both efficacy and tolerability, we utilized a mouse monoclonal antibody, MD5-1, that recognizes the mouse DR5 (mDR5) receptor. In vitro, treatment with Apo2L/TRAIL + MD5-1 enhanced cell killing in multiple mouse tumor lines, which all expressed mDR5 at similar levels. In vivo, we observed marked anti-tumor activity after treatment with Apo2L/TRAIL at 5 mg/kg for 5 days per week and MD5-1 at 1ug/mouse for 2 days per week. In the Renca model, Apo2L/TRAIL + MD5-1 treatment resulted in enhanced efficacy compared to either single agent (&amp;gt; 92% inhibition; p &amp;lt;0.0001); whereas, in the LLC1 model, an involution of the tumor tissue was observed following Apo2L/TRAIL + MD5-1 administration. On day three after treatment was initiated, a macroscopic observation of the tumors displayed a hemorrhagic appearance in both the Renca and LLC1 models. Histological analysis revealed that tumor cells, as well as, tumor-associated vasculature were positive for activated caspase 3/7 after treatment with Apo2L/TRAIL + MD5-1. Strong expression of mDR5 on both tumor cells and associated vasculature were detected, but not on vasculature in other tissues. In follow up studies, we demonstrated anti-tumor activity in a dose-dependent manner with lower doses of Apo2L/TRAIL + MD5-1 treatment. However, at higher doses, Apo2L/TRAIL + MD5-1 treatment resulted in body weight loss associated with diarrhea, dehydration, and scruffy coat. Histological analysis indicated minimal to severe apoptosis/degeneration/necrosis in the epithelium of tissues, predominantly in the gastrointestinal tract, which expressed the highest levels of mDR5. Conclusion: Our preclinical findings provide evidence that a therapeutic index can be achieved for Apo2L/TRAIL in combination with a DR5 agonist antibody and may be an effective strategy for cancer therapy. Citation Format: Timothy Sullivan, Daniel Branstetter, Pamela Holland, Tammy L. Bush. The combination of Apo2L/Trail and a death receptor 5 agonist antibody leads to enhanced antitumor activity in mouse syngeneic tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5429. doi:10.1158/1538-7445.AM2014-5429

BMP4 inhibits breast cancer metastasis by blocking myeloid-derived suppressor cell activity.

The TGFβ growth factor family member BMP4 is a potent suppressor of breast cancer metastasis. In the mouse, the development of highly metastatic mammary tumors is associated with an accumulation of myeloid-derived suppressor cells (MDSC), the numbers of which are reduced by exogenous BMP4 expression. MDSCs are undetectable in naïve mice but can be induced by treatment with granulocyte colony-stimulating factor (G-CSF/Csf3) or by secretion of G-CSF from the tumor. Both tumor-induced and G-CSF-induced MDSCs effectively suppress T-cell activation and proliferation, leading to metastatic enhancement. BMP4 reduces the expression and secretion of G-CSF by inhibiting NF-κB (Nfkb1) activity in human and mouse tumor lines. Because MDSCs correlate with poor prognosis in patients with breast cancer, therapies based on activation of BMP4 signaling may offer a novel treatment strategy for breast cancer. Cancer Res; 74(18); 5091-102. ©2014 AACR.

Tumor cell‐expressed SerpinB2 is present on microparticles and inhibits metastasis

Expression of SerpinB2 (plasminogen activator inhibitor type 2/PAI-2) by certain cancers is associated with a favorable prognosis. Although tumor-associated host tissues can express SerpinB2, no significant differences in the growth of a panel of different tumors in SerpinB2−/− and SerpinB2+/+ mice were observed. SerpinB2 expression by cancer cells (via lentiviral transduction) also had no significant effect on the growth of panel of mouse and human tumor lines in vivo or in vitro. SerpinB2 expression by cancer cells did, however, significantly reduce the number of metastases in a B16 metastasis model. SerpinB2-expressing B16 cells also showed reduced migration and increased length of invadopodia-like structures, supporting the classical view that that tumor-derived SerpinB2 is inhibiting extracellular urokinase. Importantly, although SerpinB2 is usually poorly secreted, we found that SerpinB2 effectively reaches the extracellular milieu on the surface of 0.5–1 μm microparticles (MPs), where it was able to inhibit urokinase. We also provide evidence that annexins mediate the binding of SerpinB2 to phosphatidylserine, a lipid characteristically exposed on the surface of MPs. The presence of SerpinB2 on the surface of MPs provides a physiological mechanism whereby cancer cell SerpinB2 can reach the extracellular milieu and access urokinase plasminogen activator (uPA). This may then lead to inhibition of metastasis and a favorable prognosis.

Powerful Inhibition of Experimental Human Pancreatic Cancers by Receptor Targeted Cytotoxic LH-RH analog AEZS-108

Pancreatic carcinoma is one of the cancers with the worse prognosis, thus any therapeutic improvement is imperative. Cytotoxic LH-RH analog, AN-152 (proprietary designation, AEZS-108), consisting of doxorubicin (DOX) conjugated to D-Lys⁶LH-RH, is now in clinical trials for targeted therapy of several sex hormone-dependent tumors that express LH-RH receptors. We investigated LH-RH receptors in human pancreatic carcinoma and the effects of AN-152 (AEZS-108) on experimental pancreatic cancers. We determined LH-RH receptor presence in human pancreatic cancer samples by immunohistochemistry and, in three human pancreatic cancer lines (SW-1990, Panc-1 and CFPAC-1), by binding assays and Western blotting. The effects of the cytotoxic LH-RH analog were investigated on growth of these same cancer lines xenografted into nude mice. We also analyzed differences between the antitumor effects of the cytotoxic analog and its cytotoxic radical alone, doxorubicin (DOX), on the expression of cancer-related genes by PCR arrays. LH-RH receptors were expressed in two randomly selected surgically removed human pancreatic cancer samples and in all three cancer lines. Cytotoxic LH-RH analogs powerfully inhibited growth of all three tumor lines in nude mice; AN-152 was significantly stronger than DOX on Panc-1 and CFPAC-1 cancers. PCR array showed that cytotoxic LH-RH analog AN-152 affected the expression of genes associated with cellular migration, invasion, metastasis and angiogenesis more favorably than DOX, however the changes in gene expression varied considerably among the three cancer lines. Cytotoxic LH-RH analog, AEZS-108, may be a useful agent for the treatment of LH-RH receptor positive advanced pancreatic carcinoma.

Abstract 1111: Preclinical evaluation of CP868,596, a novel PDGFRα Inhibitor for treatment of glioblastoma

Abstract Background: Recent large scale genomic studies have shown that ∼13% of glioblastoma (GBM) have a copy number gain of the gene encoding for platelet-derived growth factor receptor alpha (PDGFRα). Animal studies have shown that PDGFRα signaling can drive tumor growth. Clinical studies, however, have failed to show efficacy when patients with recurrent GBM were treated with non-specific PDGFR inhibitors (Imatinib, Sunitinib). Drawing firm conclusions from such studies is limited by lack of patient stratification by PDGFRα expression and lack of biological data demonstrating effective target inhibition. CP-868,596 is a benzimidazole which is orally bioavailable, and has IC50s of 0.9 nM and 1.8 nM for PDGFRα and PDGFRb, respectively. Phase I trials of CP-868,596 have shown a favorable safety profile and serum concentrations up to 2000 nM. We are pursuing a systematic in vitro and in vivo preclinical evaluation to assess potential clinical efficacy of CP-868,596 for the treatment of glioma. Methods: To evaluate IC50 for CP868,596, we have genetically engineered conditional Ink4a/Arf-/- mouse astrocytes to stably co-express both the human PDGFRα and its cognate ligand, PDGF-AA. Expression levels of PDGFRα and PDGF-AA levels were adjusted so they are comparable to those we have found in GBM tumors. Results and Discussion: Immunoblot analysis shows that PDGFRα phosphorylation and its downstream signaling pathway (pAKT, phospho-S6 ribosomal protein, and p-4EBP1) activated either by autocrine and/or exogenous PDGF-AA ligand (1um) is completely blocked by CP868,596 (concentrations 0.1-1um). INK4a/Arf-/-, PDGFRα/PDGF-AA astrocytes, when implanted into the NOD-SCID mouse brain or subcutaneous space (flank), form orthotopic /heterotopic tumors, respectively. Using these glioma models, intratumoral CP868,596 levels and the ability of the drug to block PDGFRα phosphorylation is being evaluated (dose range 0.1-5 ug/kg/hr IV). While multiple signal transduction pathways are implicated in pathogenesis of GBM, CP868,596 may only be effective in those GBM where PDGFR is the dominant ‘driver’ of tumorigenesis. To test this hypothesis we have established a panel of primary GBM orthotopic mouse tumor lines which overexpress PDGFRα/PDGF-AA with and without EGFR and/or cMet gene overexpression and will evaluate response to treatment with CP868,596. In addition, a Phase II treatment trial in adult glioma patients is being initiated. Data from the in vitro and in vivo studies will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1111. doi:10.1158/1538-7445.AM2011-1111

Abstract 2939: Leukocyte chemoattractant chemerin as a novel immunotherapeutic agent

Abstract Cancer immunotherapy offers the potential of potent anti-tumor responses while avoiding non-specific toxicities associated with traditional cytotoxic chemotherapy. Localization of key immune cells to tumor sites has been shown to be important for anti-tumor efficacy. Chemerin is a recently described chemoattractant initially isolated from human inflammatory fluids. Chemokine-like receptor 1 (CMKLR1) constitutes the main cellular receptor for chemerin, and is expressed by macrophages, natural killer (NK) cells, and immature dendritic cells (DCs), but has not been shown on T lymphocytes. Chemerin is synthesized and circulates systemically as an inactive precursor, prochemerin, but can be rapidly converted into its active form by proteolytic cleavage of carboxy-terminal residues by multiple proteases. To evaluate chemerin as a potential immunotherapy, we performed intratumoral injections of recombinant active murine chemerin that resulted in significantly decreased tumor growth in a B16 melanoma as well as JC breast tumor models. Tumors lines showed no expression of CMKLR1 by flow cytometric (FACS) analysis, and treatment with exogenous recombinant murine chemerin had no effect on in vitro proliferation. Compared to control media, B16 tumor-conditioned serum-free media was able to attract CMKLR1-transfected L1.2 cells (L1.2/CMKLR1) after the addition of inactive, recombinant murine prochemerin, indicating the ability of tumor-derived factors to activate prochemerin. Treatment with the pan-matrix metalloproteinase (MMP) inhibitor GM6001 resulted in reduced chemotaxis, suggesting the activation of chemerin was in part mediated by MMPs. This supports the ability of tumors to activate prochemerin. B16, as well as additional mouse tumor lines (JC, CT26, EMT6), were then transfected to constitutively express chemerin. Experiments using all tumor lines showed consistent and significantly decreased growth of chemerin-expressing tumors compared with control tumors. There were greater numbers of leukocytes in both the tumors and draining lymph nodes compared to controls. CMKLR1 knockout mice we generated showed abrogated response to chemerin-expressing tumors, supporting the hypothesis that the anti-tumor effects are mediated by this receptor. CD4 and CD8 T lymphocytes from tumor-bearing mice were isolated and shown, for the first time, to have significant CMKLR1 expression compared to control lymphocytes from naïve mice. Incubation of naïve T cells with tumor-conditioned media recapitulated these results, suggesting CMKLR1 expression can be induced by tumor-derived factors. These data suggest not only that tumor-derived proteases can activate chemerin, but also that chemerin expression at tumor sites results in the recruitment of CMKLR1-positive cells, potentially including CMKLR1+ lymphocytes, and establishment of an effective anti-tumor immune response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2939.

Regulation of proto-oncogene transcription, cell proliferation, and tumorigenesis in mice by PSF protein and a VL30 noncoding RNA

We describe the role of PSF protein and VL30-1 RNA, a mouse retroelement noncoding RNA, in the reversible regulation of proto-oncogene transcription, cell proliferation, and tumorigenesis in mice. The experiments involved increasing expression of PSF or VL30-1 RNA in NIH/3T3 fibroblast cells and B16F10 melanoma cells by transfecting the respective coding genes under control of a strong promoter or decreasing expression by transfecting a shRNA construct that causes degradation of PSF mRNA or VL30-1 RNA. The results are as follows: (i) PSF binds to the proto-oncogene Rab23, repressing transcription, and VL30-1 RNA binds and releases PSF from Rab23, activating transcription; (ii) increasing expression of PSF or decreasing expression of VL30-1 RNA suppresses cell proliferation in culture and tumorigenesis in mice; and (iii) decreasing expression of PSF or increasing expression of VL30-1 RNA promotes cell proliferation in culture and tumorigenesis in mice. These results indicate that PSF is a major tumor-suppressor protein and VL30-1 RNA is a major tumor-promoter RNA in mice. Although VL30-1 RNA can integrate into the cell genome, tumor promotion by VL30-1 RNA involves a trans effect rather than a cis effect on gene transcription. Expression of VL30-1 RNA is 5- to 8-fold higher in mouse tumor lines than in mouse fibroblast or myoblast lines, whereas expression of PSF mRNA does not decrease in the tumor lines, suggesting that tumorigenesis is driven by an increase of VL30-1 RNA rather than a decrease of PSF. A similar regulatory mechanism functions in human cells, except that human PSF-binding RNAs replace VL30-1 RNA, which is not encoded in the human genome. We propose that PSF protein and PSF-binding RNAs have a central role in the reversible regulation of mammalian cell proliferation and tumorigenesis and that increasing PSF expression or decreasing PSF-binding RNA expression in tumor cells is a potential therapeutic strategy for cancer.

Comparative epigenomics of human and mouse mammary tumors

Gene silencing by aberrant epigenetic chromatin alteration is a well-recognized event contributing to tumorigenesis. Although genetically engineered tumor-prone mouse models have proven a powerful tool in understanding many aspects of carcinogenesis, to date few studies have focused on epigenetic alterations in mouse tumors. To uncover epigenetically silenced tumor suppressor genes (TSGs) in mouse mammary tumor cells, we conducted initial genome-wide screening by combining the treatment of cultured cells with the DNA demethylating drug 5-aza-2'-deoxycytidine (5-azadC) and the histone deacetylase inhibitor trichostatin A (TSA) with expression microarray. By conducting this initial screen on EMT6 cells and applying protein function and genomic structure criteria to genes identified as upregulated in response to 5-azadC/TSA, we were able to identify two characterized breast cancer TSGs (Timp3 and Rprm) and four putative TSGs (Atp1B2, Dusp2, FoxJ1 and Smpd3) silenced in this line. By testing a panel of 10 mouse mammary tumor lines, we determined that each of these genes is commonly hypermethylated, albeit with varying frequency. Furthermore, by examining a panel of human breast tumor lines and primary tumors we observed that the human orthologs of ATP1B2, FOXJ1 and SMPD3 are aberrantly hypermethylated in the human disease whereas DUSP2 was not hypermethylated in primary breast tumors. Finally, we examined hypermethylation of several genes targeted for epigenetic silencing in human breast tumors in our panel of 10 mouse mammary tumor lines. We observed that the orthologs of Cdh1, RarB, Gstp1, RassF1 genes were hypermethylated, whereas neither Dapk1 nor Wif1 were aberrantly methylated in this panel of mouse tumor lines. From this study, we conclude that there is significant, but not absolute, overlap in the epigenome of human and mouse mammary tumors.

Tes, a Specific Mena Interacting Partner, Breaks the Rules for EVH1 Binding

The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

Quantitative Estimates of Vascularity in Solid Tumors by Non-Invasive Near-Infrared Spectroscopy

We examined the relationship between non-invasive estimates of the tumor hemoglobin concentration by near-infrared spectroscopy (NIBS) and histological scores of tumor vascularity by Chalkley counts in seven tumor lines in nude mice [malignant gliomas: U87, U118, U373; small cell lung cancers (SCLC): 54A, 5413, DMS79; prostate cancer: MatLyLu (MILL) ]. We also evaluated the effect of continuous anti-angiogenic treatment with TNP-470 on tumor hemoglobin concentration and tumor vascularity in U87 and MILL tumors. Non-invasive LAIRS recordings were performed with a custom-built flash near-infrared spectrometer using light guidecoupled reflectance measurements at 800±10 nm. Chalkley counts were obtained from CD31-immunostained cryosections. The LAIRS recordings in arbitrary absorbance units increased with tumor size in the individual tumors until a plateau was reached at approximately 150 mm3. This plateau was relatively tumor line-specific. LAIRS recordings at the plateau phase were strongly correlated (P<.001, n=71) to the histological vessel score (Chalkley count) of the same individual tumors excised immediately after the LAIRS was performed. Non-invasive LAIRS recordings of the highly vascularized gliomas (U87, U118, and U373) plus the MatLyLu tumor line were significantly higher than the three less vascularized SCLC tumor lines (P<.001). Continuous treatment with the anti-angiogenic compound TNP-470, an endothelial cell inhibitor, significantly retarded tumor growth in both U87 and MILL tumors, but all tumors eventually grew. When comparing treated and untreated tumors of similar size, both LAIRS recordings and Chalkley counts were significantly lower in TNP-470-treated tumors (P<.05). In conclusion, the LAIRS technique provides a non-invasive measure of the degree of vascularization in untreated tumors and the LAIRS technique can measure modifications in tumor vascularization by anti-angiogenic therapy.

Loss of PTEN expression leading to high Akt activation in human multiple myelomas

Mouse plasma cell tumor (PCT) and human multiple myeloma (MM) are terminal B-cell malignancies sharing many similarities. Our recent work demonstrated that activation of the insulin-like growth factor receptor (IGF-IR)/insulin receptor substrate (IRS)/phosphatidylinositol 3' kinase (PI 3'K) pathway was evident in the tumor lines derived from both species. Although PI 3'K activity was higher in mouse tumor lines than that in human tumors, activation of Akt serine/threonine kinase was markedly lower in mouse lines. This discrepancy prompted us to test the status of PTEN tumor suppressor gene, as it has been shown to be a negative regulator of PI 3'K activity. Although all the mouse lines expressed intact PTEN, 2 of the 4 human lines (Delta47 and OPM2) possessing the highest Akt activity lost PTEN expression. Sequencing analysis demonstrated that the PTEN gene contains a deletion spacing from exon 3 to exon 5 or 6 in the Delta47 line and from exon 3 to 7 in the OPM2 line. Restoration of PTEN expression suppressed IGF-I-induced Akt activity, suggesting that loss of PTEN is responsible for uncontrolled Akt activity in these 2 lines. Despite the expression of PTEN with the concomitant low Akt activity in all mouse PCT lines, their p70S6K activities were generally higher than those in 3 human MM lines, arguing for specific negative regulation of Akt, but not p70S6K by PTEN. These results suggest that p70S6K and Akt may be differentially used by the plasma cell tumors derived from mice and humans, respectively.

Loss of PTEN expression leading to high Akt activation in human multiple myelomas

AbstractMouse plasma cell tumor (PCT) and human multiple myeloma (MM) are terminal B-cell malignancies sharing many similarities. Our recent work demonstrated that activation of the insulin-like growth factor receptor (IGF-IR)/insulin receptor substrate (IRS)/phosphatidylinositol 3′ kinase (PI 3′K) pathway was evident in the tumor lines derived from both species. Although PI 3′K activity was higher in mouse tumor lines than that in human tumors, activation of Akt serine/threonine kinase was markedly lower in mouse lines. This discrepancy prompted us to test the status of PTEN tumor suppressor gene, as it has been shown to be a negative regulator of PI 3′K activity. Although all the mouse lines expressed intact PTEN, 2 of the 4 human lines (Δ47 and OPM2) possessing the highest Akt activity lost PTEN expression. Sequencing analysis demonstrated that the PTEN gene contains a deletion spacing from exon 3 to exon 5 or 6 in the Δ47 line and from exon 3 to 7 in the OPM2 line. Restoration of PTEN expression suppressed IGF-I–induced Akt activity, suggesting that loss of PTEN is responsible for uncontrolled Akt activity in these 2 lines. Despite the expression of PTEN with the concomitant low Akt activity in all mouse PCT lines, their p70S6K activities were generally higher than those in 3 human MM lines, arguing for specific negative regulation of Akt, but not p70S6K by PTEN. These results suggest that p70S6K and Akt may be differentially used by the plasma cell tumors derived from mice and humans, respectively.