Tumor Cells In Mouse Model Research Articles

Abstract A035: Radiation-induced senescence as a driver of glioblastoma recurrence

Abstract Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting matrix metalloproteinases, chemokines, cytokines, and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells as well as promoted tumor cell proliferation and radioresistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and publicly available data from the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells, like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also observed in PDX models of GBM. Using these PDX models, we are currently validating novel senolytic-based therapeutic approaches to mitigate tumor recurrence after radiotherapy. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy. Citation Format: Ben R. Jordan, Nozomi Tomimatsu, Luis Macedo Di Cristofaro, Suman Kanji, Bipasha Mukherjee, Sandeep Burma. Radiation-induced senescence as a driver of glioblastoma recurrence [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A035.

Abstract A009: DNA damage-induced senescence as a driver of glioblastoma recurrence

Abstract Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and the normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting proteases, cytokines and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these cells senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells and promoted tumor cell proliferation and radiation resistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also seen in multiple PDX models of GBM. Using these PDX lines, we demonstrate that adjuvant senolytic treatment can delay or even prevent recurrence in pre-clinical mouse models of GBM. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy. Citation Format: Sandeep Burma, Nozomi Tomimatsu, Luis Cristofaro, Suman Kanji, Benjamin Jordan, Bipasha Mukherjee. DNA damage-induced senescence as a driver of glioblastoma recurrence [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A009.

Abstract PR001: DNA damage-induced senescence as a driver of glioblastoma recurrence

Abstract Glioblastomas (GBM) are treated with high doses of ionizing radiation (IR), yet these tumors inevitably recur, and the recurrent tumors are highly therapy resistant. An understanding of mechanisms driving GBM recurrence is critical for improving therapy. During GBM therapy, both the tumor and the normal brain tissue surrounding the tumor are treated with up to 60 Gy of IR. IR is a potent inducer of senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting proteases, cytokines and growth factors that create a senescence-associated secretory phenotype (SASP). We have shown previously that IR-induced senescence of astrocytes in the brain leads to the secretion of SASP factors that promote the growth and invasiveness of tumor cells in mouse models of GBM (Fletcher-Sananikone, et al, Cancer Research, 2021). Following up on this study, we irradiated a panel of GBM cell lines and found that a significant fraction of these cells senesced rapidly in a p21-dependent manner. Senescent glioma cells upregulated SASP genes in a NF-kB-dependent manner, prominently, the interleukin IL6, which is an activator of the JAK-STAT pathway. Conditioned media from senescent GBM cells activated the JAK-STAT pathway in non-senescent GBM cells and promoted tumor cell proliferation and radiation resistance. Interestingly, conditioned media from senescent GBM cells could also activate the NF-kB pathway and induce SASP in non-senescent cells. These results indicate that senescent GBM cells can activate pro-tumorigenic pathways in their non-senescent counterparts and SASP can spread from senescent to non-senescent cells. Bioinformatic analyses of the transcriptomic profiles of irradiated GBM cells and the TCGA database revealed that the inhibitor of apoptosis protein cIAP2 is a critical survival factor for senescent glioma cells. We find that targeting cIAP2 using Smac mimetics triggers apoptosis of senescent GBM cells with minimal toxicity towards normal brain cells like astrocytes. Upregulation of cIAP2 in irradiated tumor cells was also seen in multiple PDX models of GBM. Using these PDX lines, we demonstrate that adjuvant senolytic treatment can delay or even prevent recurrence in pre-clinical mouse models of GBM. In sum, our findings illustrate how senescence of both stromal and tumor cells promote GBM recurrence via different mechanisms and underscore the potential utility of adjuvant senolytic therapy for blunting GBM recurrence after radiotherapy. Citation Format: Sandeep Burma, Nozomi Tomimatsu, Luis Cristofaro, Suman Kanji, Benjamin Jordan, Bipasha Mukherjee. DNA damage-induced senescence as a driver of glioblastoma recurrence [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR001.

The spatial and single-cell analysis reveals remodeled immune microenvironment induced by synthetic oncolytic adenovirus treatment

Oncolytic viruses are multifaceted tumor killers, which can function as tumor vaccines to boost systemic antitumor immunity. In previous study, we rationally designed a synthetic oncolytic adenovirus (SynOV) harboring a synthetic gene circuit, which can kill tumors in mouse hepatocellular carcinoma (HCC) models. In this study, we demonstrated that SynOV could sense the tumor biomarkers to lyse tumors in a dosage-dependent manner, and killed PD-L1 antibody resistant tumor cells in mouse model. Meanwhile, we observed SynOV could cure liver cancer and partially alleviate the liver cancer with distant metastasis by activating systemic antitumor immunity. To understand its high efficacy, it is essential to explore the cellular and molecular features of the remodeled tumor microenvironment (TME). By combining spatial transcriptome sequencing and single-cell RNA sequencing, we successfully depicted the remodeled TME at single cell resolution. The state transition of immune cells and stromal cells towards an antitumor and normalized status exemplified the overall cancer-suppressive TME after SynOV treatment. Specifically, SynOV treatment increased the proportion of CD8+ T cells, enhanced the cell-cell communication of Cxcl9-Cxcr3, and normalized the Kupffer cells and macrophages in the TME. Furthermore, we observed that SynOV could induce distant responses to reduce tumor burden in metastatic HCC patient in the Phase I clinical trial. In summary, our results suggest that SynOV can trigger systemic antitumor immunity to induce CD8+ T cells and normalize the abundance of immune cells to remodel the TME, which promises a powerful option to treat HCC in the future.

Monitoring the Cell Cycle of Tumor Cells in Mouse Models of Human Cancer.

Cell division is obligatory to tumor growth. However, both cancer cells and noncancer cells in tumors can be found in distinct stages of the cell cycle, which may inform the growth potential of these tumors, their propensity to metastasize, and their response to therapy. Hence, it is of utmost importance to monitor the cell cycle of tumor cells. Here we discuss well-established methods and new genetic advances to track the cell cycle of tumor cells in mouse models of human cancer. We also review recent genetic studies investigating the role of the cell-cycle machinery in the growth of tumors in vivo, with a focus on the machinery regulating the G1/S transition of the cell cycle.

Abstract 4203: Tumor-targeted delivery of a neoantigen surrogate by non-pathogenic Listeria reduces metastatic ovarian cancer

Abstract Objectives: End-stage ovarian cancer is commonly characterized by intraperitoneal (IP) carcinomatosis and chemoresistance. To date, immune-mediated therapies have shown little success in this population in part due to the absence of neoantigens in these tumors and strong immune suppression in the tumor microenvironment (TME) preventing T cell activation. Our laboratory has developed a novel microbial-based immunotherapeutic strategy for selective delivery of a highly immunogenic tetanus toxoid protein (TT856-1313) into tumor cells through in vivo infection with attenuated Listeria monocytogenes. In mice previously vaccinated against TT, this leads to reactivation of pre-existing TT-specific memory T cells which kill the infected tumor cells in mouse models of pancreatic cancer. The addition of gemcitabine (GEM) improves T cell responses by reducing immune suppression in the TME. We hypothesize that the regimen Listeria-TT+GEM will have efficacy in vivo in mice with advanced ovarian cancer. Methods: Infection and killing of mouse and human ovarian tumor cells by Listeria-TT was analyzed in vitro. C57Bl/6 mice were vaccinated with the TT vaccine at six weeks of age. 3x106 luciferase-expressing ID8 syngeneic mouse tumor cells (Id8-Luc) were injected IP and tumor growth was monitored by Vivo Imaging Systems (IVIS.) Mice were divided into four treatment groups: saline, GEM, Listeria-TT, and Listeria-TT+GEM. Quantification of tumor burden was approximated by total flux (photons/second) within a region of interest (ROI) prior to, during, and at completion of therapy (days 0, 9, and 19 after tumor cell injection, respectively). Results: In vitro analyses demonstrated efficient infection and killing of mouse Id8-Luc and chemoresistant human Hey ovarian tumor cells by Listeria-TT. The response of tumors and metastases to the four prespecified treatment regimens was evaluated by IVIS as described above. On Day 0, there was no significant difference in the quantified tumor burden between the four treatment groups. On Days 9 and 19, the tumor burden was significantly less in the Listeria-TT+GEM group compared to the saline group (Mann-Whitney p=0.0159). Neither GEM nor Listeria-TT alone were significantly different from the saline group on day 9, but the effect of GEM was significantly different from saline on day 19 (p=0.0079). In a follow up study, the survival time of the Listeria-TT+GEM-treated mice was improved by 50% compared to the saline mice. The survival of GEM compared to saline is under investigation. Conclusions: Our results demonstrate that Listeria-TT+GEM strongly reduced metastatic ovarian cancer in mice. Additional experiments are ongoing to evaluate if more treatment cycles of Listeria-TT+GEM further improves the survival time. This study suggest that the combination therapy may be promising for human application. Citation Format: Lisa R. Gabor, Olivia R. Khouri, Ken Lin, Nicole Nevadunsky, Dennis Yi-Shin Kuo, Claudia Gravekamp. Tumor-targeted delivery of a neoantigen surrogate by non-pathogenic Listeria reduces metastatic ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4203.

Differential Localization and Invasion of Tumor Cells in Mouse Models of Human and Murine Leukemias.

Leukemias are refractory hematopoietic malignancies, for which the development of new therapeutic agents requires in vivo studies using tumor-bearing mouse models. Although several organs are commonly examined in such studies to evaluate the disease course, the effectiveness of interventions and the localization of tumor cells in the affected organs are still unclear. In this study, we histologically examined the distribution of leukemia cells in several organs using two leukemic mouse models produced by the administration of two cell lines (THP-1, a human myelomonocytic leukemia, and A20, a mouse B cell leukemia/lymphoma) to severe immunodeficient mice. Survival of the mice depended on the tumor burden. Although A20 and THP-1 tumor cells massively infiltrated the parenchyma of the liver and spleen at 21 days after transplantation, A20 cells were hardly found in connective tissues in Glisson’s capsule in the liver as compared with THP-1 cells. In the bone marrow, there was more severe infiltration of A20 cells than THP-1 cells. THP-1 and A20 cells were widely spread in the lungs, but were rarely observed in the small intestine. These findings suggest that each leukemia model has a unique localization of tumor cells in several affected organs, which could critically affect the disease course and the efficacy of therapeutic agents, including cellular immunotherapies.

Application of Alphaviral Vectors for Immunomodulation in Cancer Therapy.

The lack of specific and efficient cancer therapies has influenced the development of novel approaches, such as immunotherapy, which from its original application of immunogenic protein delivery has developed into the use of more sophisticated recombinant gene delivery methods to achieve better safety and efficacy profiles. This approach involves viral and non-viral delivery systems. Expression vectors have been engineered for alphaviruses, including Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus. For immunotherapeutic applications, recombinant particles, RNA replicons and layered DNA vectors that express tumor-associated antigens (TAAs) and cytokines have been studied in animal models and in a few clinical trials. Immunization studies with TAAs and cytokines have elicited strong antibody responses and vaccination has provided protection against challenges with tumor cells in mouse models. Furthermore, the combination of TAAs and cytokines, antibodies and growth factors and the co-administration of chemotherapeutics and bacteriabased adjuvants have enhanced immunogenicity. Intratumoral and systemic delivery of recombinant alphavirus particles has demonstrated significant tumor regression and prolonged survival rates in rodent tumor models. Alphavirus-based immunotherapy represents a rapid and efficient method for prophylactic and therapeutic applications in animal models.

MDA-MB-231 breast cancer cells fuel osteoclast metabolism and activity: A new rationale for the pathogenesis of osteolytic bone metastases

Recent progress in dissecting the molecular paracrine circuits of cancer and stromal cells in bone metastases (BM) are offering new options to improve current merely palliative approach. The study of tumor-stroma metabolic interplay may further ameliorate this scenario. In this context, we demonstrated that highly glycolytic MDA-MB-231 cancer cells, that form osteolytic BM in vivo, release a large amount of lactate at a significantly higher level than MCF7 cells. Thus, we speculated that lactate released from carcinoma cells is uptaken and metabolically used by osteoclasts, the key players of osteolysis associated with BM. First, we demonstrated that the release of lactate at the bone site is mediated by monocarboxylate transporter 4 (MCT4), as revealed by immunostaining and MCT4 localization at the plasma membrane of tumor cells in mouse model of BM and in human tissue sections of BM. Then, we showed that in vitro lactate is uptaken by osteoclasts to be used as a fuel for the oxidative metabolism of osteoclasts, ultimately enhancing Type I collagen resorption. The passive transport of lactate into osteoclasts was mediated by MCT1: MCT1 expression is significantly upregulated during osteoclast differentiation and Type I collagen resorption is significantly impaired when osteoclasts are treated with 7-(N-benzyl-N-methylamino)-2-oxo-2H-chromene-3-carboxylic acid, an MCT-1 inhibitor. Together, these data demonstrate that lactate released by glycolytic breast carcinoma cells in the bone microenvironment promotes the formation of osteolytic lesions, and provide the rationale for further studies on the use of MCT1 targeting as a novel therapeutic approach in advanced cancer patients with BM.

Abstract 2822: Establishing the use of Vortex technology for investigating circulating tumor cells in mouse models of breast cancer

Abstract Background: Circulating tumor cells (CTCs) are precursors of metastatic disease and are an important indicator of the disease progression and outcome of many cancers. Though several recent studies have advanced our understanding of CTCs, many critical areas of CTC biology remain largely unexplored. Murine models of cancer offer a unique opportunity to address this issue. However, small sample volumes, low number of CTCs, difficulty to access ports for blood collection are challenges that diminish the utility of mouse models for studying CTCs. In our previous studies, we had validated the use of Vortex technology for the label-free capture of CTCs from blood samples of different cancers. In this current study, we now report our recent, unpublished data on the adaptation of Vortex platform for enriching and characterizing murine and human tumor cells from mouse blood. Methods: By spiking tumor cells into small volumes of mouse blood (200-400 µl) from cardiac puncture, we tested the impact of i) different dilutions of mouse blood (10X, 20X, 40X); ii) varying numbers of tumor cells; iii) different cell types (MDA-MB-231, 4T1 and EMT6); and iv) sample recycling on capture efficiency and purity. Blood was processed through the Vortex plastic chip and, to accurately identify and enumerate the enriched tumor cells, we developed a staining protocol that labels human- or murine-specific cytokeratin, vimentin, and CD45. Results: Our results reveal that over a range of dilutions tested, just after one cycle, 10X dilution of mouse blood was superior, yielding a capture efficiency of 38.83% and a purity of 33% for MDA-MB-231 cells, compared to 20X (34.16% efficiency, 31.1% purity), and 40X (31.83% efficiency, 31% purity) dilutions. We are currently testing whether recycling the blood flow-through would increase the recovery. Even when as many as ~7500 tumor cells were spiked in mouse blood, the Vortex platform was able to successfully enrich tumor cells with high consistency and purity. Importantly, we were now able to successfully isolate human and murine tumor cell lines with varying levels of epithelial cell adhesion molecule (EpCAM) expression (4T1 - high, MDA-MB-231 - moderate to low) spiked into mouse blood. Experiments confirming the viability and growth rate of cancer cells isolated from mouse blood using MTT assay will also be presented. Conclusion: In summary, our results reveal the use of the Vortex technology for studying CTCs in murine models of cancer, with the ability to handle volumes of blood as low as 200uL while having a capacity to capture extremely large number of CTCs. While this study was focused on breast cancer, our workflow is easily adapted to other malignancies. Studies are underway to grow viable CTCs from tumor bearing animals and isolated by this platform for subsequent growth in 3D culture systems for future biologic and drug testing studies. Citation Format: Vishnu Ramani, Rakhi Gupta, Melanie Triboulet, Corinne Renier, Steve C. Crouse, Elodie Sollier-Christen, Stefanie S. Jeffrey. Establishing the use of Vortex technology for investigating circulating tumor cells in mouse models of breast cancer [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 2822. doi:10.1158/1538-7445.AM2017-2822

A novel intracellular antibody against the E6 oncoprotein impairs growth of human papillomavirus 16-positive tumor cells in mouse models.

Single-chain variable fragments (scFvs) expressed as “intracellular antibodies” (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. Here we use an intrabody targeting the E6 oncoprotein of Human papillomavirus 16 (HPV16) to address the issue of a non-invasive therapy for HPV cancer patients.A scFv against the HPV16 E6 was selected by Intracellular Antibody Capture Technology and expressed as I7nuc in the nucleus of HPV16-positive SiHa, HPV-negative C33A and 293T cells. Colocalization of I7nuc and recombinant E6 was observed in different cell compartments, obtaining evidence of E6 delocalization ascribable to I7nuc. In SiHa cells, I7nuc expressed by pLNCX retroviral vector was able to partially inhibit degradation of the main E6 target p53, and induced p53 accumulation in nucleus. When analyzing in vitro activity on cell proliferation and survival, I7nuc was able to decrease growth inducing late apoptosis and necrosis of SiHa cells.Finally, I7nuc antitumor activity was demonstrated in two pre-clinical models of HPV tumors. C57BL/6 mice were injected subcutaneously with HPV16-positive TC-1 or C3 tumor cells, infected with pLNCX retroviral vector expressing or non-expressing I7nuc. All the mice injected with I7nuc-expressing cells showed a clear delay in tumor onset; 60% and 40% of mice receiving TC-1 and C3 cells, respectively, remained tumor-free for 17 weeks of follow-up, whereas 100% of the controls were tumor-bearing 20 days post-inoculum. Our data support the therapeutic potential of E6-targeted I7nuc against HPV tumors.

Methods to study primary tumor cells and residual tumor cells in mouse models of oncogene dependence.

The studies of oncogene dependence are aimed to understand an unfortunate and puzzling aspect of targeted anticancer treatments-their progression to drug resistance. Drug resistance develops from a pool of cells that survive the original treatment, called minimal residual disease. Mouse models based on tetracycline-dependent expression of transgenic oncogenes are used to imitate targeted oncogene blockade and to reproduce minimal residual disease in humans. Here we describe a novel method for generating oncogene-dependent mammary tumors using somatic transfer of transactivator-containing retroviruses into transgenic mice with tetracycline-dependent oncogenes and a method for measuring continuous mitotic activity in epithelial cells in real time.

Human breast cancer and lymph node metastases express Gb3 and can be targeted by STxB-vectorized chemotherapeutic compounds.

BackgroundThe B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen.MethodsUsing a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model.ResultsGb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p ≤ 0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p ≤ 0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p ≤ 0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.ConclusionThe enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.

Abstract 2367A: Understanding the role of stem-like tumor cells in mouse models of high-grade gliomas

Abstract High-grade gliomas (WHO grade III-IV) are typified by poor therapeutic response and rapid lethality. Since the discovery of glioma cells with properties reminiscent of neural stem cells, there has been a great deal of interest in understanding their contributes to tumor growth and therapeutic resistance. Self-renewal is the defining characteristic of both normal and cancer stem cells, and it has been hypothesized that glioma cells with self-renewal capacity contribute disproportionately to tumor growth compared to other, more differentiated lineages. However, analyses are complicated by the lack of reliable markers to definitively identify and functionally characterize each distinct cell lineage. In the normal neurogenic niche, ID (Inhibitor of DNA-binding) genes regulate self-renewal and multipotency of adult neural stem cells. Here, using mouse models of high-grade glioma driven by PDGF- or mutKRAS, we show that high Id1 expression (Id1high) identifies self-renewing, multipotent tumor cells. By contrast, low Id1 expression (Id1low) identifies progenitor-like cells with proliferative potential but limited self-renewal capacity. While Id1high cells generate tumors upon orthotopic transplantation, Id1low cells do so with shorter latency and higher penetrance. Further, eliminating tumor cell self-renewal through targeted deletion of ID genes has modest effects on overall survival, while knockdown of the bHLH transcription factor, Olig2, within the Id1low population, results in a significant survival benefit. Together, these data highlight an important distinction between self-renewal capacity and tumor growth potential in two mouse models of gliomagenesis, underscoring the importance of non-stem cell lineages in disease progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2367A. doi:1538-7445.AM2012-2367A

Detection and quantification of circulating tumor cells in mouse models of human breast cancer using immunomagnetic enrichment and multiparameter flow cytometry

Circulating tumor cells (CTCs) in the peripheral blood of breast cancer patients may be an important indicator of metastatic disease and poor prognosis. However, the use of experimental models is required to fully elucidate the functional consequences of CTCs. The purpose of this study was to optimize the sensitivity of multiparameter flow cytometry for detection of human tumor cells in mouse models of breast cancer. MDA-MB-468 human breast cancer cells were serially diluted in whole mouse blood. Samples were lysed and incubated with a fluorescein isothiocyanate-conjugated anti-human leukocytic antigen antibody and a phycoerythrin-conjugated anti-mouse pan-leukocyte CD45 antibody. Samples were then immunomagnetically depleted of CD45-positive leukocytes, fixed, permeabilized, and stained with propidium iodide before flow cytometric analysis. Human breast cancer cells could be differentiated from mouse leukocytes based on increased light scatter, cell surface marker expression, and aneuploid DNA content. The method was found to have a lower sensitivity limit of 10(-5) and was effective for detecting human breast cancer cells in vivo in the circulation of experimental mice carrying primary human mammary tumors. This technique has the potential to be a valuable and sensitive tool for investigating the biological relevance of CTCs in experimental mouse models of breast cancer.