Cancer Cell Line Xenograft Models Research Articles

Abstract 4656: Novel mitochondria-based targeting overcomes resistance in prostate cancer

Abstract Background: Prostate cancer (PC) is a major contributor to cancer-related mortality in men, with metastatic castration-resistant PC (mCRPC) presenting a significant obstacle due to its resistance to standard treatments. Current therapies, including nonsteroidal antiandrogens and chemotherapy drugs, provide limited effectiveness and are often accompanied by severe side effects. Recently, statins have gained attention as antitumor agents, demonstrating chemopreventive effects on a range of human cancers while maintaining a well-established safety profile. However, the use of statins as standalone treatment is limited by insufficient cancer cell targeting. Methods: We synthesized a DZ-simvastatin (DZ-SIM) conjugate that combines a near-infrared heptamethine carbocyanine dye (DZ) with simvastatin for selective tumor cell targeting. Cancer cell survival was measured by crystal violet and MTT assay. Co-localization of DZ-SIM with mitochondria and lysosome in cells was detected under confocal microscope using Mitotracker and Lysotracker. Cellular and mitochondrial cholesterol levels were detected by LC-MS. Male nude mice were inoculated subcutaneously with 22Rv1-Luc cells as a prostate cancer xenograft mouse model. We used Swiss-PDB and Rosetta algorithms to model the structure of the OATP1B3 channel. Oxygen consumption rate and extracellular acidification rate were measured by Seahorse assay. After treatment with DZ-SIM, differentially expressed genes profiling of prostate cancer cells was performed using RNAseq. Results: Our research demonstrates the conjugate's ability to effectively kill androgen-independent and metastatic PC cells without harming normal cells. Furthermore, DZ-SIM has been shown to kill multiple human PC cell lines, regardless of androgen receptor status or therapeutic resistance. DZ-SIM has been shown to inhibit growth in human prostate cancer cell line xenograft models. NIR fluorescence imaging showed specific localization of DZ-SIM to cancer, but not to normal cells and tissues. It could localize into cancer cells by binding the transmembrane transporter OATP to primarily target subcellular organelle mitochondria, decreasing the OCR and ECAR of cancer cells. DZ-SIM can bind to multiple cytosolic and mitochondrial proteins as detected by NIRF imaging. Cholesterol levels also decrease in whole cells and mitochondria. Ingenuity Pathway Analysis (IPA) of RNAseq showed that the four signaling pathways in the DZ-SIM treatment group are significantly enriched in genes related to cholesterol synthesis. Conclusion: DZ-SIM specifically targets PC cells and their mitochondria, resulting in loss of mitochondrial function and consequent cell death. With a unique subcellular targeting strategy, the conjugate holds the potential to outperform existing chemotherapeutic drugs. Citation Format: Yan Ou, Adrian Lim, Ning Zhai, Mouad Edderkaoui, Stephen Pandol, Ruoxiang Wang, Yi Zhang. Novel mitochondria-based targeting overcomes resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4656.

Targeting LRRC15 Inhibits Metastatic Dissemination of Ovarian Cancer.

Dissemination of ovarian cancer (OC) cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine-rich repeat-containing membrane protein, highly overexpressed in OC bowel metastases compared to matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of OC demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with β1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody-drug conjugate ABBV-085 in both early and late metastatic OC cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived OC xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting OC metastasis and suggest a novel and promising therapy to target OC metastases.

A High-Content Screen for C/EBPα Expression Identifies Novel Therapeutic Agents in Dedifferentiated Liposarcoma.

Dedifferentiated liposarcoma (DDLS), one of the most common and aggressive sarcomas, infrequently responds to chemotherapy. DDLS survival and growth depend on underexpression of C/EBPα, a tumor suppressor and transcriptional regulator controlling adipogenesis. We sought to screen and prioritize candidate drugs that increase C/EBPα expression and may therefore serve as differentiation-based therapies for DDLS. We screened known bioactive compounds for the ability to restore C/EBPα expression and inhibit proliferation selectively in two DDLS cell lines but not in normal adipose-derived stem cells (ASC). Selected hits' activity was validated, and the mechanism of the most potent, SN-38, was investigated. The in vivo efficacy of irinotecan, the prodrug of SN-38, was evaluated in DDLS xenograft models. Of 3,119 compounds, screen criteria were met by 19. Validation experiments confirmed the DDLS selectivity of deguelin, emetine, and SN-38 and showed that they induce apoptosis in DDLS cells. SN-38 had the lowest IC50 (approximately 10 nmol/L), and its pro-apoptotic effects were countered by knockdown of CEBPA but not of TP53. Irinotecan significantly inhibited tumor growth at well-tolerated doses, induced nuclear expression of C/EBPα, and inhibited HIF1α expression in DDLS patient-derived and cancer cell line xenograft models. In contrast, doxorubicin, the most common treatment for nonresectable DDLS, reduced tumor growth by 30% to 50% at a dose that caused weight loss. This high-content screen revealed potential treatments for DDLS. These include irinotecan, which induces apoptosis of DDLS cells in a C/EBPα-dependent, p53-independent manner, and should be clinically evaluated in patients with advanced DDLS.

Synergistic Activity of Paclitaxel, Sorafenib, and Radiation Therapy in advanced Renal Cell Carcinoma and Breast Cancer

Advanced cancer has been shown to be associated with a higher percentage of epigenetic changes than with genetic mutations. Preclinical models have shown that the combination of paclitaxel, sorafenib, and radiation therapy (RT) plays a crucial role in renal cell carcinoma (RCC) and breast cancer. This study aimed to investigate the involvement of mitochondrial cytochrome c–dependent apoptosis in the mechanism of action of a combination of paclitaxel, sorafenib, and RT in RCC and breast cancer. RCC and breast cancer cell lines were exposed to paclitaxel and sorafenib alone or combined in the presence of radiation, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The synergistic anticancer effects of the combination therapy on cell cycle and intracellular signaling pathways were estimated using flow cytometry and immunoblot analysis. RCC and breast cancer cell line xenograft models were used to examine the antitumor activity in vivo. Our results suggest that paclitaxel, sorafenib, and RT synergistically decreased the viability of RCC and breast cancer cells and significantly induced their apoptosis, as shown by caspase-3 cleavage. Paclitaxel, sorafenib, and radiation cotreatment reduced antiapoptotic factor levels in these cells and, thereby, significantly reduced the tumor volume of RCC and breast cancer cell xenografts. The current study suggests that paclitaxel, sorafenib, and radiation cotreatment was more effective than cotreatment with paclitaxel or sorafenib and radiation. These findings may offer a new therapeutic approach to RCC and breast cancer.

Preparation and anti-cancer evaluation of promiximab-MMAE, an anti-CD56 antibody drug conjugate, in small cell lung cancer cell line xenograft models

Antibody-drug conjugates (ADCs) have been successfully applied clinically as target drugs for cancer. In this study, anti-neural cell adhesion molecule also called CD56 antibody-monomethyl auristatin E (MMAE) conjugate named Promiximab-MMAE was prepared by conjugation of microtubule inhibitor MMAE with Promiximab. The average drug-to-antibody ratio (DAR) of Promiximab-MMAE was 3.13 as analysed by liquid chromatography–mass spectrometry/ mass spectrometry (LC-MS/MS). The targeting capacity and affinity kinetics of Promiximab-MMAE were similar to that of Promiximab after being conjugated with MMAE as tested by flow cytometry and biolayer interferometry analysis. Promiximab-MMAE showed effective anti-proliferation on CD56-positive cell lines (NCI-H524, NCI-H526, and NCI-H69), with the half maximal inhibitory concentration (IC50) values of 19.24, 5.23, and 0.32 nmol/L in vitro, respectively. Promiximab-MMAE of 10 mg/kg every three days with a total of three times was administered in vivo. Results showed that the tumour regression was not recrudesced in NCI-H69 and NCI-H526 xenograft mice models till 52 and 56 days. Moreover, body weight and histopathology of the major organs (liver, spleen, heart, lung, and kidney) showed no significant changes after treatment with Promiximab-MMAE. In conclusion, Promiximab-MMAE is a potential candidate for the treatment of CD56 positive small cell lung cancer.

1639P - Tucatinib, a HER2 selective kinase inhibitor, is active in patient derived xenograft (PDX) models of HER2-amplified colorectal, esophageal and gastric cancers

Background: Tucatinib, an orally bioavailable and HER2 selective small molecule tyrosine kinase inhibitor, is currently being developed for HER2+ metastatic breast cancer in combination with capecitabine + trastuzumab (HER2CLIMB study). In addition to breast cancer, HER2 is amplified in subsets of patients with other malignancies, including gastrointestinal cancers (colorectal, esophageal and gastric cancers). To test whether tucatinib might have utility in treating HER2-amplified cancers originating from the gastrointestinal tract, tucatinib was tested alone, and in combination with trastuzumab, in cell line derived and PDX models of colorectal, esophageal and gastric cancer. Methods:In vitro assays were performed to evaluate the combination of tucatinib and trastuzumab in HER2-amplified cell lines by measuring changes in signal transduction (pHER2, pHER3, pAKT) and cell survival. The in vivo activity of tucatinib (50 mg/kg BID) and trastuzumab (20 mg/kg Q3D) was evaluated alone, or in combination, in established HER2-amplified tumor models, including PDX models of colorectal, esophageal and gastric cancers. Results: As a single agent, or in combination with trastuzumab, tucatinib demonstrated significant anti-tumor activity, including tumor regressions, in the N87 gastric cancer cell line xenograft model and in PDX models of HER2 amplified colorectal, esophageal and gastric cancers. The combination of tucatinib and trastuzumab was consistently more active than either single agent alone, and resulted in tumor growth inhibition from 85-139%, including complete tumor regressions in HER2+ gastric PDX models. Conclusions: The activity of tucatinib in HER2-amplified colorectal, esophageal and gastric tumor xenograft models supports the exploration of using tucatinib to treat HER2+ gastrointestinal cancers in the clinical setting. To this end, an open label phase II clinical study combining tucatinib with trastuzumab in HER2+/RAS wild type metastatic colorectal cancer (MOUNTAINEER) has recently been initiated. Legal entity responsible for the study: Cascadian Therapeutics Funding: Cascadian Therapeutics Disclosure: S. Peterson: Employee and shareholder of Cascadian Therapeutics, corporate officer of Cascadian Therapeutics. P. de Vries, J. Piasecki, R. Rosler: Employee and shareholder of Cascadian Therapeutics

Abstract 129: Assessing the mechanism and therapeutic potential of modulators of the human mediator complex-associated protein kinases CDK8 and CDK19

Abstract Mediator-associated protein kinases CDK8 and CDK19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We identified two structurally differentiated chemical series, suitable for exploring their function. In addition to tools that fulfil the criteria set out for chemical probes, the lead compounds from each series, CCT251921 and MSC2530818, had optimal pharmacological and pharmaceutical properties making them suitable for preclinical studies. Having potent, highly selective, orally bioavailable exemplar compounds from these series in hand, we were well positioned to investigate the therapeutic potential of dual CDK8/19 inhibition. The compounds exhibited modest anti-tumor activity in colorectal cancer cell line xenograft models with modulation of p-STAT1SER727, a target engagement biomarker, and altered gene expression profiles, including super-enhancer regulated gene expression, consistent with the inhibition of CDK8/19. In PDX-derived cell cultures we observed inhibition of soft-agar growth in cells derived from different tumor types. However, we only detected significant antitumour activity in 1 of 6 colorectal PDX models tested in vivo, and one example of sensitization to standard of care chemotherapy, despite showing inhibition of p-STAT1SER727. Acute myeloid leukemia cells were the most sensitive cancer type in the PDX panel with therapeutic potency seen in systemic and sub-cutaneous models. Significantly, the compounds impacted on stem cell biology. In a bone progenitor model we saw dose-responsive activation and inhibition of markers of bone matrix and bone deposition that was distinct from WNT blockade. Treatment of a diverse collection of normal cell co-culture models detected a unique response profile consistent with stimulation of an immune/inflammatory response. In vivo treatment of a genetically engineered mouse model expressing oncogenic beta-catenin shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. Finally, in pre-clinical tolerability studies we observed a similar, widespread adverse safety profile at therapeutically relevant exposures for both CCT251921 and MSC2530818. At the concentrations tested we detected >80% inhibition of p-STAT1SER727 and increased IL-12 plasma levels. Since the observed pathological effects were generated with two potent, highly selective, but structurally distinct compounds, we conclude that the adverse consequences of treatment are the direct result of inhibition of CDK8 and/or CDK19. The serious and complex nature of the toxicity observed indicates that the clinical development of either series of CDK8/19 modulators, or other chemotypes with similar profiles, will be extremely challenging. Citation Format: Paul A. Clarke, Maria-Jesus Ortiz-Ruiz, Robert Te Poele, Olajumoke Adeniji-Popoola, Gary Box, Christina Esdar, Kenneth Ewan, Sharon Gowan, Alexis De Haven Brandon, Phllip Hewitt, Wolfgang Kaufmann, Aurelie Mallinger, Florence Raynaud, Felix Rohdich, Kai Schiemann, Stephanie Simon, Richard Schneider, Melanie Valenti, Julian Blagg, Trevor Dale, Suzanne Eccles, Paul Workman, Dirk Wienke Dirk Wienke. Assessing the mechanism and therapeutic potential of modulators of the human mediator complex-associated protein kinases CDK8 and CDK19 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 129. doi:10.1158/1538-7445.AM2017-129

HDAC and PD-1 inhibition in humanized triple-negative breast cancer xenografts.

e14604 Background: There is great interest in investigating immune checkpoint inhibition in combination with novel agents, however, preclinical studies are limited by the lack of immune-competent human cancer cell line xenograft models. We developed a “hematopoietic” humanized mouse model to address this unmet need. HDAC inhibitors (HDACi) induce T-cell chemokine expression and enhance response to PD1 inhibitors in lung cancer models. We evaluated the combination of OKI-179, a novel Class I HDACi and nivolumab in our humanized mouse model. Methods: BRGS newborn pups were transplanted with CD34+ cells purified from umbilical cord blood. At 16 weeks, MDA-MB-231 cells were implanted in the flanks of the mice. When the average tumor size reached ~150-300 mm3, the mice were randomized into vehicle, nivolumab, OKI-179, or the combination treatment according to % chimerism. At the end of the treatment, mice were euthanized and tissues were collected for further analysis. Results: We observed a statistically significant improvement in tumor growth inhibition with the combination of OKI-179 and nivolumab (%TGI = 77%) as compared to single agent nivolumab (45%TGI). Nivolumab treatment led to a significant decrease in PD-1 expression on T cells. We also observed an increased number of TILs (CD8+ T cells) and T cells in the lymph nodes of treated mice suggesting T cell expansion. All mice were highly chimeric and responding tumors exhibited an increase in CD44 high IFN+ T cells. We observed a higher CD8% and a higher effector memory % (HLADR+, CD45RO+) in these mice. Conclusions: We successfully established a humanized TNBC human cancer cell line xenograft model, with tumor engraftment occurred in all humanized mice. Mice treated with nivolumab demonstrating the development of lymph nodes that were populated by activated T cells. The combo resulted in superior tumor growth inhibition. These preliminary results demonstrate that human immunity and PD-1 expressing T cells exist in these models and provide the basis for planned immunotherapy combination studies.

Abstract C126: PRLR ADC: A novel antibody drug-conjugate for the treatment of PRLR positive breast cancer

Abstract Breast cancer is a heterogeneous disease comprised of various subtypes based on pathology and molecular profiling. Expression of hormone receptors (HR) and HER2 biomarkers are important determinants of therapy choice, due to the established role of these proteins as drivers of disease. Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed on a subset of breast cancers and may contribute to pathogenesis. Functionally, activation of PRLR by the hormone ligand Prolactin (PRL) induces PRLR dimerization and signaling resulting in cell proliferation and differentiation. While PRLR is expressed at low levels in some normal human tissues including the mammary gland, it is relatively overexpressed in ∼25% of human breast tumors and importantly, is rapidly internalized upon binding of anti-PRLR antibodies. We developed an anti-PRLR antibody-drug conjugate (ADC), PRLR ADC, to target PRLR positive breast cancer. PRLR ADC is comprised of a fully human high affinity function-blocking anti-PRLR IgG1 antibody conjugated via a non-cleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated anti-PRLR antibody and the PRLR ADC block PRL mediated activation in vitro and induce rapid internalization of the receptor into lysosomes. PRLR ADC induces potent cell cycle arrest and cytotoxicity in several PRLR-expressing cell lines. The in vivo efficacy of PRLR ADC was explored in breast cancer cell line xenograft models expressing both endogenous PRLR (MCF7, T47D) or transfected receptor (MCF7/PRLR). Treatment of tumor bearing SCID (T47D) or NCr Nude (MCF7) animals was initiated approximately 15 days post implantation of cells where tumor volumes averaged 150-200 mm3. In both T47D and MCF7/PRLR xenograft models, where PRLR is expressed highly, single or multiple (once weekly x 3) doses of 2.5-15 mg/kg resulted in significant inhibition of tumor xenograft growth. In the MCF7 model that expresses low levels of PRLR, inhibition and regression of tumors was observed at 10 and 15 mg/kg dose levels. In all models, higher doses resulted in greater and more prolonged repression of tumor growth. Conjugation of DM1 to anti-PRLR antibody was required for efficacy, as unconjugated antibody had no effect on tumor growth. Anti-tumor efficacy of PRLR ADC was also assessed in NSG mice bearing breast cancer Patient Derived Xenograft (PDXs) tumors with moderate and heterogeneous expression of PRLR. Treatment was initiated 21 days after implantation of the PDX tumors where the average tumor volume was ∼500mm3. Anti-tumor efficacy was observed following 10 or 20 mg/kg PRLR ADC dosed once weekly x 4. These studies demonstrate the promising anti-tumor activity of the PRLR ADC against PRLR positive breast cancers and support the continued development of this agent. Citation Format: Marcus P. Kelly, Sandra Coetzee, Carlos Hickey, Sosina Makonnen, Frank Delfino, Julian Andreev, Arthur Kunz, Christopher D'Souza, Jason Giurleo, Thomas Nittoli, Pamela A. Trail, Nicholas Papadopoulos, Gavin Thurston, Jessica R. Kirshner. PRLR ADC: A novel antibody drug-conjugate for the treatment of PRLR positive breast cancer. [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 C126.

Abstract 2312: Identifying novel cancer stem cell target for triple-negative breast cancer

Abstract Triple negative breast cancer is characterized by its aggressive clinical behavior with high incidence of metastasis to the lungs and brain. These patients do not respond to hormonal therapy and show intrinsic resistance to conventional chemotherapy. Robust evidence indicates that treatment-resistance and metastases may arise from a subpopulation of cells with tumor-initiating capacity called breast cancer stem cells (BCSC). We are one of the first groups to demonstrate that residual tumors after exposure to chemotherapy are enriched for BCSC. We have previously described a treatment-resistant gene signature of 493 genes derived from patient biopsies.This finding narrowed down the possible cancer stem cell growth related genes from genome wide to only 493 genes and provided candidate genes for screening potential targets that affect BCSC self-renewal. Re-evaluation of the candidate genes determined a previously unidentified cancer gene, Hematological and Neurological Expressed 1-Like (HN1L) as the target of cancer stem cell self-renewal. The objective of this study was to investigate the role of HN1L in regulating BCSC and metastasis in TNBC, and to determine the mechanism of action of HN1L in BCSC. Knocking down HN1L by shRNA in SUM159 and MDAMB231 cell lines significantly decreased mammosphere forming efficiency (MSFE) and CD44+/CD24low/- population. To assess the contribution of HN1L to BCSC and tumor growth, a patient derived human-cancer-in-mouse xenograft model and two cancer cell line xenograft models were employed. To ensure targeted delivery, siRNA was packaged into DOPC liposomes and delivered into mice via intraperitoneal injection. Results showed that silencing HN1L alone or in combination with chemotherapy in xenografts reduced tumor volume and BCSC population as measured by MSFE, CD44+/CD24low/- markers, ALDF+ cell population and limiting dilution assay. Liposomal HN1L siRNA treated mice also showed lower level of lung metastasis. A gene signature obtained from microarray analysis on HN1L siRNA treated tumor tissues highly correlated with better prognosis in TNBC patients. Overexpression of HN1L in TNBC cell lines activated STAT3 signaling, induced CD44+/CD24low/- population and cancer cell migration. Moreover, chemoresistance were also observed with HN1L overexpression. HN1L colocalized with CNTFR and STAT3 in cytoplasm and nucleus by immunofluorescence staining. Changes in HN1L expression level resulted in corresponding changes in phospho-STAT3. Furthermore, CNTFR knockdown compromised the effect of HN1L overexpression in activating STAT3.These results suggest that the HN1L gene plays an important role in BCSC through regulating CNTFR-STAT3 signaling pathway, and is associated with poor prognosis. Taken together, the results from our study demonstrate for the first time that HN1L is a novel target for TNBC. Targeting HN1L and/or CNTFR-STAT3 pathway might provide an alternative therapeutic strategy for TNBC. Citation Format: Yi Liu, Dong Soon Choi, Sergio Grandos-Principal, Wei Qian, Lacey Burey, Helen Wong, Crisitian Rodriguez-Aguayo, Anil Sood, Zheng Li, Stephen Wong, Heidi Weiss, Bhuvanesh Dave, Melissa Landis, Jenny C. Chang. Identifying novel cancer stem cell target for triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2312. doi:10.1158/1538-7445.AM2015-2312

Abstract 3240: Understanding clonal complexity of a tumor xenograft model via cellular barcoding technology

Abstract Recent advances in next-generation sequencing have revealed the presence of genetic heterogeneity and clonal evolution within tumors. Intratumoral heterogeneity has been implicated in the disease progression, metastasis, therapeutic responses and development of drug resistance. Although cancer cell line xenograft models have been extensively used in cancer research to test drug efficacy, it is unknown how much clonal heterogeneity is maintained in the process of cell line xenograft establishment. In order to quantitatively assess the clonal complexity in xenograft models, here we applied a cellular barcoding technology using the HCC827 cell line. HCC827 is a non-small cell lung cancer cell line containing an exon 19 deletion which has been shown to be clinically relevant due to its initial response to EGFR inhibitors followed by resistance. This barcoding tool allowed us to label each individual cell with one unique DNA barcode via lentiviral infection and monitor the clonal heterogeneity within the implanted cell population by quantifying the number of unique barcodes. We were able to estimate the percentage of implanted clones that actually contributed to the formation of cell line xenografts. This study provides valuable insight on the clonal diversity present in xenograft models, which will further elucidate the heterogeneous nature of tumors. Citation Format: Justina X. Caushi, Hyo-eun C. Bhang, Jie Li, Iris Kao, Viveksagar Krishnamurthy Radhakrishna, Vesselina G. Cooke, Joshua M. Korn, David A. Ruddy, Shailaja Kasibhatla, Frank Stegmeier. Understanding clonal complexity of a tumor xenograft model via cellular barcoding technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2015-3240

Tumor Imaging and Interferon-γ–Inducible Protein-10 Gene Transfer Using a Highly Efficient Transferrin-Conjugated Liposome System in Mice

We have developed a PEGylated transferrin-conjugated liposomes (PTf-Ls) system for the combined tumor imaging and targeted delivery of the IFN-γ-inducible protein-10 (IP-10) gene in a single macromolecular construct. Here, we characterize and analyze the use of this system in a mouse model of breast cancer. The biophysical and cell transfection properties of PTf-Ls were determined through a series of in vitro experiments. A nude mouse/breast cancer cell line xenograft model (mouse xenograft model) was used to image the tumor internalization of fluorescently labeled PTf-Ls. The clinical use of the system was tested by treating tumor-bearing mice with PTf-Ls loaded with IP-10 plasmid DNA or fluorescent lipoplexes. The resulting 165-nm liposomes (zeta potential = -10.6 mV) displayed serum resistance, low cytotoxicity (<5%), and high transfection efficiency (≤82.8%) in cultured cells. Systemic intravenous administration of fluorescent PTf-Ls in the mouse xenograft model resulted in nanoparticle circulation for 72 hours, as well as selective and efficient internalization in tumor cells, according to in vivo fluorescence and bioluminescence analyses. Tumor fluorescence increased gradually up to 26 hours, whereas background fluorescence decreased to near-baseline levels. Treatment of mice with PTf-Ls entrapped pcDNA3.1-IP-10 suppressed tumor growth in mice by 79% on day 50 and increased the mean survival time of mice. Fluorescent pcDNA-IP-10-entrapped PTf-Ls showed good properties for simultaneous tumor-targeted imaging and gene-specific delivery in an animal tumor model. Our developed transferrin-conjugated liposome system possesses promising characteristics for tumor-targeting, imaging, and gene therapy applications.

Abstract 3726: Combinations of HDAC inhibitor, chemotherapeutic agent and retinoic acid induce growth arrest, differentiation and tumor regression in preclinical models of breast cancer.

Abstract The histone deacetylase inhibitor (HDACi), entinostat, is being actively explored as a new-generation epigenetic drug which can lead to the change in the expression status of genes/pathways, but has low efficacy in cancer monotherapy. All-trans retinoic acid (ATRA) induces the differentiation of various types of stem cells. Data from cell culture and xenograft models from our lab showed that a combination of entinostat (MS-275), doxorubicin and ATRA effectively decreased tumor size in three breast cancer cell line xenograft models. Here, we sought to further investigate the mechanism of action of the triple drug combination in cancer cells; in particular, its effect on the breast cancer stem cell population. We performed a comprehensive genome wide analysis of gene expression of MDA-MB-231 breast cancer cells treated with ATRA, MS-275 and doxorubicin as monotherapies and as combination therapies. We saw that the drug-response gene profile of ATRA is very similar to DMSO (vehicle)-treated cells. Accordingly, the addition of ATRA (A) to MS-275 (MA), Dox (AD) and MS-275/Dox (MAD) displayed minimal changes in the gene expression profile of each of the other treatments. Addition of Dox to MS-275 (MD), on the other hand, potentiated the “reprograming” effect of MS-275 and affected the expression of many antitumor genes known to be related to cell cycle and growth arrest. It also altered expression of genes involved in development and inflammation. The most differentially expressed genes, validated by qPCR, were novel genes from the cancer/testis antigens and tripartite motif (TRIM) family of proteins. Interestingly, in MDA-MB-231and SUM149 cells, even the addition of low doses of doxorubicin (12.5 nM) to MS-275 increased 2 and 2.6 fold the G2 cell cycle arrest in comparison to Dox and MS-275, respectively. Despite the gene expression pattern similarity between MS-275/Dox (MD) and MS-275/Dox/ATRA (MAD) groups, we saw that MAD was more effective in inducing cell death and apoptosis in vitro and in vivo. The epithelium specific ETS transcription factor-1 (ESE-1) was differentially regulated between MAD and MD and is, in fact, part of the MS-275/ATRA (MA) signature. Using limiting dilution transplantation assays in mammary fat pads of immunodeficient mice we observed that MAD treatment in vivo most effectively targeted breast cancer stem cells (BCSC) compared to any other combination of drugs. The cancer stem cell frequency of the cells isolated from MAD treated mice was 1 in 236,570. The second most effective treatment for BCSC was MA (1 in 150,721), followed by ATRA&amp;gt; MS-275&amp;gt;MD&amp;gt;Dox&amp;gt;DMSO&amp;gt;AD. In conclusion, the reprogramming events initiated by HDACi and retinoid sensitize the cells to low doses of doxorubicin. The combination therapy may have a significant effect in decreasing breast tumor growth and recurrence. Citation Format: Vanessa F. Merino, Nguyen Nguyen, Helen Sadik, Sean Cho, Xian Chong Zhou, Qian Chen, Duojia Pan, Saraswati Sukumar. Combinations of HDAC inhibitor, chemotherapeutic agent and retinoic acid induce growth arrest, differentiation and tumor regression in preclinical models of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3726. doi:10.1158/1538-7445.AM2013-3726