Patient-derived Breast Tumor Xenografts Research Articles

Abstract 1921: Stem-like clusters of CD44+ circulating tumor cells seed metastases in breast cancer

Abstract Background: Circulating tumor cells (CTCs) are cells shed from primary tumor and circulate in the peripheral blood. They are considered the seeds of metastasis. Compared to single CTCs, clusters of multiple CTCs possess 50 times higher metastatic capacity in mouse breast cancer models. However, the mechanisms underlying the metastatic promotion effect of CTC clusters are unclear. In addition, whether clustered CTCs have cancer stem cell (CSC) properties and what stem cell markers they express have not been determined. Methods: Immunohistochemistry (IHC) was used to detect CTCs in vascular structures, and the stem cell markers in single and clustered CTCs. Lung metastases were either monitored by bioluminescence imaging (BLI) or visualized by fluorescence microscopy. Stem cell properties were examined by mammosphere assays in vitro and tumorigenic assays in vivo. Clustering assay was performed by culturing cells in Poly-HEMA coated plates (for cell lines) or collagen-coated plates (for primary cells derived from patient-derived breast tumor xenografts, PDXs), and then monitored by IncuCyte live cell dynamic imaging. Results: CD44+ enriched circulating tumor cell (CTC) clusters were found in the lung/liver vascular structures in vivo in both metastatic breast cancer patients and PDXs that develop spontaneous lung metastases. Comparing to single CD44+ cells, clustered CD44+ cells formed more mammospheres, increased tumorigenic potential, and promoted lung colonization. Combining siRNA-mediated knockdown and CRISPR/Cas-based knockdout, we found that CD44 is required for breast tumor cell cluster formation and lung colonization upon tail vein seeding. During cluster formation, EGFR was activated, which improved both stemness and survival of clustered CTCs. Anti-EGFR antibody mimicked CD44 knockdown to inhibit cluster formation of PDX-derived tumor cells. Administration of EGFR inhibitor Erlotinib efficiently inhibited CD44+ cells-mediated spontaneous metastases to the lungs without affecting primary tumor growth. Conclusions: Our data provide new insights into the cellular and molecular mechanisms of stem-like clustered CD44+ cells-seeded metastasis, and implicate that targeting CD44+ CTC clusters by inhibition of EGFR activity could be a new therapeutic strategy to treat metastatic breast cancer. Citation Format: Xia Liu, Wenjing Chen, Rokana Taftaf, Huiping Liu. Stem-like clusters of CD44+ circulating tumor cells seed metastases in 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 1921. doi:10.1158/1538-7445.AM2017-1921

Integrated Bottom-Up and Top-Down Proteomics of Patient-Derived Breast Tumor Xenografts

Bottom-up proteomics relies on the use of proteases and is the method of choice for identifying thousands of protein groups in complex samples. Top-down proteomics has been shown to be robust for direct analysis of small proteins and offers a solution to the “peptide-to-protein” inference problem inherent with bottom-up approaches. Here, we describe the first large-scale integration of genomic, bottom-up and top-down proteomic data for the comparative analysis of patient-derived mouse xenograft models of basal and luminal B human breast cancer, WHIM2 and WHIM16, respectively. Using these well-characterized xenograft models established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium, we compared and contrasted the performance of bottom-up and top-down proteomics to detect cancer-specific aberrations at the peptide and proteoform levels and to measure differential expression of proteins and proteoforms. Bottom-up proteomic analysis of the tumor xenografts detected almost 10 times as many coding nucleotide polymorphisms and peptides resulting from novel splice junctions than top-down. For proteins in the range of 0–30 kDa, where quantitation was performed using both approaches, bottom-up proteomics quantified 3,519 protein groups from 49,185 peptides, while top-down proteomics quantified 982 proteoforms mapping to 358 proteins. Examples of both concordant and discordant quantitation were found in a ∼60:40 ratio, providing a unique opportunity for top-down to fill in missing information. The two techniques showed complementary performance, with bottom-up yielding eight times more identifications of 0–30 kDa proteins in xenograft proteomes, but failing to detect differences in certain posttranslational modifications (PTMs), such as phosphorylation pattern changes of alpha-endosulfine. This work illustrates the potency of a combined bottom-up and top-down proteomics approach to deepen our knowledge of cancer biology, especially when genomic data are available.

Preclinical Efficacy of Ron Kinase Inhibitors Alone and in Combination with PI3K Inhibitors for Treatment of sfRon-Expressing Breast Cancer Patient-Derived Xenografts.

Recent studies have demonstrated that short-form Ron (sfRon) kinase drives breast tumor progression and metastasis through robust activation of the PI3K pathway. We reasoned that upfront, concurrent inhibition of sfRon and PI3K might enhance the antitumor effects of Ron kinase inhibitor therapy while also preventing potential therapeutic resistance to tyrosine kinase inhibitors (TKI). We used patient-derived breast tumor xenografts (PDX) as high-fidelity preclinical models to determine the efficacy of single-agent or dual Ron/PI3K inhibition. We tested the Ron kinase inhibitor ASLAN002 with and without coadministration of the PI3K inhibitor NVP-BKM120 in hormone receptor-positive [estrogen receptor (ER)(+)/progesterone receptor (PR)(+)] breast PDXs with and without PIK3CA gene mutation. Breast PDX tumors harboring wild-type PIK3CA showed a robust response to ASLAN002 as a single agent. In contrast, PDX tumors harboring mutated PIK3CA demonstrated partial resistance to ASLAN002, which was overcome with addition of NVP-BKM120 to the treatment regimen. We further demonstrated that concurrent inhibition of sfRon and PI3K in breast PDX tumors with wild-type PIK3CA provided durable tumor stasis after therapy cessation, whereas discontinuation of either monotherapy facilitated tumor recurrence. Our work provides preclinical rationale for targeting sfRon in patients with breast cancer, with the important stipulation that tumors harboring PIK3CA mutations may be partially resistant to Ron inhibitor therapy. Our data also indicate that tumors with wild-type PIK3CA are most effectively treated with an upfront combination of Ron and PI3K inhibitors for the most durable response. Clin Cancer Res; 21(24); 5588-600. ©2015 AACR.

Abstract 1514: Acquired differentiation and loss of malignancy of pulmonary metastases in breast cancer

Abstract Cancer stem cells (CSCs) or tumor initiating cells (TICs) were demonstrated in a leukemia transplant experiment in 1930s with a single mouse leukemic cell as well as in recent studies of human leukemia and solid tumors. Our work demonstrated that breast CSCs are able to mediate spontaneous lung metastasis in patient-derived breast tumor xenografts (PDXs) in vivo (PNAS, 2010). We further characterized the phenotypic and molecular characteristics of spontaneous metastases versus primary breast tumors. We collected the tumor cells from the lungs and conducted orthotopic implantation back into the mouse mammary fat pads in order to make a more metastatic tumor model. Surprisingly, relative to the parental primary breast tumors, the lung metastasis (met)-derived mammary tumors exhibited a slower growth rate and a reduced metastatic potential with a more differentiated epithelial status based on gene array analyses. The up-regulated genes in the met-derived tumors included promoters of cell adhesion and differentiation as well as tumor suppressors and cell cycle inhibitors, such as KRT19 (CK19), CEACAM1 (CD66a), and CDKN1A. Many of the down-regulated genes were promoters of self-renewal, cell motility, and cell cycle, such as EZH2, SMAD2, and CCNB1. Clinically, this signature correlated with breast tumor subtypes. Consistently, the differentiation scores of the lung met-derived tumors were significantly higher than the primary tumors (p = 0.007). We then examined the functions of microRNA candidates in regulating CSC-mediated metastasis and therapy resistance as well as their clinical relevance. By miRNA analyses, thirty-two miRNAs were differentially expressed with twenty-seven miRNAs being up-regulated in the met-derived tumors, including miR-200 family members which induce epithelial phenotype and inhibits self-renewal, miR-30 family members, miR-138, and others. Further clinical studies demonstrated that miR-30c is independently associated with favorable distant relapse free survival of breast tumor patients (GSE22216 n = 210). Subsequent mechanistic work characterized that miR-30c inhibits both CSC-mediated metastasis and chemotherapy resistance by a direct targeting of cytoskeleton genes twinfilin TWF1 and vimentin (VIM). We also identified miR-138 as a novel regulator of invasion and epithelial-mesenchymal transition in breast cancer cells, acting by directly targeting the polycomb epigenetic regulator EZH2. Notably, based on the UNC breast tumor database analyses (n = 337), the miR-138 activity signature served as a novel independent prognostic marker for patient survival beyond traditional pathologic variables, intrinsic subtypes or a proliferation gene signature. Both miR-30c and miR-138 can be regulated by the transcription factor GATA3. Our results highlight the loss of malignant character in some lung micro-metastatic lesions and the epigenetic regulation of this phenotype. Note: This abstract was not presented at the meeting. Citation Format: Huiping Liu. Acquired differentiation and loss of malignancy of pulmonary metastases in 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 1514. doi:10.1158/1538-7445.AM2015-1514

Abstract PL-2: Patient Derived Xenografts - Pre-Clinical Models for Prevention and Treatment of Metastasis

Abstract Twenty to thirty percent of breast cancer patients eventually develop metastasis despite receiving state-of-the-art therapies. Clearly, identifying better ways to eliminate micro-metastatic tumor cells is paramount to eradicating death from breast cancer. Unfortunately, history suggests that developing yet another drug to target yet another pathway involved in tumor progression will not cure breast cancer. We now know that every tumor is unique; each tumor is heterogenous; tumors hijack multiple, redundant pathways to survive and grow; dormant tumor cells are resistant to chemotherapy; and tumors evolve resistance under pressure of therapy. To better understand the problem of metastasis, and to develop better treatments, we must utilize in vivo models of metastasis that best recapitulate heterogenous human breast cancers. We have generated a collection of orthotopic patient-derived breast tumor xenograft (PDX) models from all of the major breast cancer subtypes. We found remarkable fidelity between PDX models and bona fide breast tumors, including the ability to metastasize and to evolve under pressure of treatment. This presentation will highlight the strengths and pitfalls of PDX models of breast cancer and how they might be utilized to develop new strategies for treatment. Citation Format: Alana L. Welm. Patient Derived Xenografts - Pre-Clinical Models for Prevention and Treatment of Metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PL-2.

Validation of p27KIP1 expression levels as a candidate predictive biomarker of response to rapalogs in patient-derived breast tumor xenografts.

Blockade of mammalian target of rapamycin (mTOR) is a promising area in breast cancer therapy. However, in clinical trials, objective response rate with mTOR inhibitor monotherapy in breast cancer was modest. Biomarker studies designed to identify the responders of rapalogs are of increasing interest. We validated p27KIP1 expression levels as a candidate predictive biomarker of response to rapalogs. We also analyzed the correlation between rapamycin activity and p27KIP1 expression in the primary breast cancer cells and the patient-derived breast tumor xenograft models. The cells isolated from the breast tumor tissues expressing high levels of p27KIP1 were sensitive to rapamycin, whereas the cells from the tissues expressing low levels of p27KIP1 exhibited resistance to rapamycin. The correlation between p27KIP1 expression and rapamycin antitumor activity was also observed in the patient-derived breast tumor xenograft models. Moreover, we also found rapamycin significantly decreased phosphorylated p70S6K1 and phosphorylated 4EBP1 in both samples. It seemed that the different sensitivity of tumor cells to rapamycin did not owe to its different potency against mTOR activity. We further propose p27KIP1 expression level may be also a candidate predictive biomarker of rapalogs for breast cancer therapy, which requires additional clinical validation.

Abstract A33: Combined therapy of a novel anti-NOTCH2/3 antibody with paclitaxel inhibits tumor growth in a patient-derived breast tumor xenograft

Abstract Cancer stem cells (CSCs) have been identified as the small subpopulation of cells in many cancers and are thought to be responsible for cancer initiation, progression, metastasis, recurrence and drug resistance. The NOTCH pathway, comprised of ligands JAG1, JAG2, DLL1, DLL3 and DLL4 and receptors NOTCH1, NOTCH2, NOTCH3 and NOTCH4, plays a critical role in stem cell signaling, cell proliferation, survival, apoptosis, and differentiation. Because of the existing body of data that implicates aberrant NOTCH pathway activation in resistance to chemotherapeutic agents in epithelial and hematologic tumors, we hypothesized that NOTCH inhibition would enhance the anti-tumor activity of chemotherapeutic agents. We have identified a NOTCH3 activating mutation in a patient breast cancer and developed a xenograft tumor model from the primary patient sample, OMP-B37. The combination of paclitaxel and OncoMed's anti-Notch2/3 antibody, OMP-59R5, inhibited the tumor growth of OMP-B37 xenograft tumors. In addition, we found that treatment with OMP-59R5 drove changes in tumor heterogeneity as indicated by the increased expression of markers associated with a mature cellular fate. The down-regulation of tumor growth promoting genes and NOTCH pathway markers was observed with OMP-59R5 treatment. The enhanced activity of combined OMP-59R5 and paclitaxel therapy in patient-derived tumor xenografts suggests that there is therapeutic potential in combining targeted NOTCH pathway inhibition with chemotherapeutic agents. Citation Format: Jie Wei, Jennifer Cain, Jalpa Shah, Breanna Wallace, Tracy Tang, Min Wang, Christopher Murriel, Cancilla Belinda, Austin Gurney, Aaron Sato, John Lewicki, Ann Kapoun, Tim Hoey. Combined therapy of a novel anti-NOTCH2/3 antibody with paclitaxel inhibits tumor growth in a patient-derived breast tumor xenograft. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A33.

Comprehensive quantitative analysis of ovarian and breast cancer tumor peptidomes.

Aberrant degradation of proteins is associated with many pathological states, including cancers. Mass spectrometric analysis of tumor peptidomes, the intracellular and intercellular products of protein degradation, has the potential to provide biological insights on proteolytic processing in cancer. However, attempts to use the information on these smaller protein degradation products from tumors for biomarker discovery and cancer biology studies have been fairly limited to date, largely due to the lack of effective approaches for robust peptidomics identification and quantification and the prevalence of confounding factors and biases associated with sample handling and processing. Herein, we have developed an effective and robust analytical platform for comprehensive analyses of tissue peptidomes, which is suitable for high-throughput quantitative studies. The reproducibility and coverage of the platform, as well as the suitability of clinical ovarian tumor and patient-derived breast tumor xenograft samples with postexcision delay of up to 60 min before freezing for peptidomics analysis, have been demonstrated. Moreover, our data also show that the peptidomics profiles can effectively separate breast cancer subtypes, reflecting tumor-associated protease activities. Peptidomics complements results obtainable from conventional bottom-up proteomics and provides insights not readily obtainable from such approaches.

Abstract 3909: Assessing the role of tumor vascularity in nanotherapeutics delivery.

Abstract Chemotherapeutic delivery is generally poor in tumors characterized by rapid perfusion and low blood volume fraction. Nanoparticles can be engineered to overcome abnormal flow conditions to act as intravascular drug depots for the localized delivery of high concentrations of chemotherapeutics. We hypothesized that the ability of multi-stage nanotherapeutics to accumulate in tumors is highly dependent on tumor perfusion and that matching particle parameters to tumor-specific flow parameters can improve the delivery of nanotherapeutics. To test this hypothesis, we measured first-pass perfusion of an intra-arterial bolus of FITC-dextran using intravital microscopy (IVM) in 4 patient-derived breast tumor xenograft (PDX) lines: BCM-2665, BCM-4195, BCM-2147, and BCM-3887. These PDX lines were established directly from patient samples and maintain the triple negative biomarker status of the original patient tumors. Selected for their differing vascular morphologies, these 4 tumor lines yielded distinct, reproducible transport features unique to each tissue. BCM-2665 tumors (n=6) were characterized by regions of dense vascularization interspersed with regions of little to no blood flow. BCM-2147 tumors (n=6) were dominated by large tortuous vessels like those observed in BCM-2665 tumors, but also had a well-developed capillary network interconnecting the large vessels. BCM-3887 tumors (n=6) were characterized by an extensive network of poorly defined, dilated, leaky microvessels. BCM-4195 tumors were uniformly covered with a well-defined, widely spaced microvessel network. The arteriovenous transit time (AVTT) was measured by tracking a bolus of 40kDa FITC-dextran through the tumor vasculature in real-time. Both 2665 and 2147 tumors were rapidly perfused (∼10 - 25 seconds), whereas a majority of 3887 and 4195 tumors required ∼45 seconds or more. The blood volume fraction (BVF) varied by nearly an order of magnitude, with 2665 and 2147 tumors exhibiting the lowest BVF (∼0.11 - 0.12). The tracer elimination constant (Kel), which was measured as a surrogate for tumor permeability using time-lapse IVM images, was an order of magnitude lower for 3887 and 4195 tumors (∼0.11/hr vs. ∼1.17/hr) indicating that these tumors are much more permeable than 2665 and 2147 tumors. Interestingly, flow parameters that adversely impact drug accumulation appear to favor particle accumulation. In clinical studies, tumors characterized by rapid perfusion and low blood volume fraction generally respond poorly to chemotherapy and radiation therapy. We have observed this phenomenon in our cancer models: BCM-2665 and BCM-2147 tumors fail to show growth inhibition at clinically relevant doses of docetaxel, whereas BCM-4195 and BCM-3887 tumors show complete growth inhibition. As such, we believe these tumors will provide an excellent model for studying the relative importance of tumor-specific transport features for improving drug delivery. Citation Format: Anne L. Van de Ven-Moloney, Melissa D. Landis, Lacey A. Burey, Mauro Ferrari, Jenny C. Chang. Assessing the role of tumor vascularity in nanotherapeutics delivery. [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 3909. doi:10.1158/1538-7445.AM2013-3909

Abstract 1398: Establishment and characterization of a clinically-relevant personalized hormone-dependent breast cancer tumor model.

Abstract Background: The START Patient Tumor Program was established to improve clinical outcomes for cancer patients by creating a biorepository of blood and cancer tissues for characterization and preclinical model development and testing. To date samples from over 1500 patients have been collected with over 400 models generated including 40 patient-derived breast tumor xenografts. These models can be used for personalized studies, particularly in situations where treatment options have been exhausted. In the current study a breast biopsy was collected from a 48 year old Hispanic female with recurrent, chemotherapy-refractory ER+/HER2+ metastatic breast cancer and implanted into an immune-deficient mouse. The resulting model was expanded and evaluated against eight FDA-approved and four investigational agents. Methods: ST340 ER+/HER2+ breast PDX model: The tumor sample from an inform-consented participant was implanted into nude mice and the established model confirmed by histologic analysis and linked with clinical treatment and outcome data. Activating mutation profiling was performed on fifty-two known cancer genes with extracted DNA using a PCR-based assay with customized primers. An efficacy study was performed testing various approved and investigational agents. Study endpoints included tumor volume and time from treatment initiation. Reported results included tumor growth inhibition or delay and regression. Results: Prior to treatment initiation, estradiol was withheld to confirm dependence and was found requisite for growth. Following treatment, results identified impressive sensitivity to the targeted therapy everolimus including tumor regressions and moderate sensitivity towards an aromatase inhibitor. Treatment with this combination regimen resulted in improved tumor markers and overall quality of life for an extended period of time. Following progression, subsequent treatment with a cytotoxic agent found active in the initial screen resulted in additional months of progression free disease. Conclusion: Participation in the START Patient Tumor Program provided beneficial data which improved clinical outcome for this patient demonstrating that personalized preclinical models of human cancer can predict useful agents for clinical treatment in select situations. Citation Format: Michael J. Wick, Teresa L. Vaught, Lizette Gamez, Jennifer Brown, Alyssa Moriarty, Anthony W. Tolcher, Drew W. Rasco, Amita Patnaik, Gladys Rodriguez, Ronald L. Drengler, Kyriakos P. Papadopoulos. Establishment and characterization of a clinically-relevant personalized hormone-dependent breast cancer tumor model. [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 1398. doi:10.1158/1538-7445.AM2013-1398

Patient-derived breast tumor xenografts facilitating personalized cancer therapy

Despite improved detection and reduction of breast cancer-related deaths over the recent decade, breast cancer remains the second leading cause of cancer death for women in the US, with 39,510 women expected to succumb to metastatic disease in 2012 alone (American Cancer Society, Cancer Facts &Figures 2012. Atlanta: American Cancer Society; 2012). Continued efforts in classification of breast cancers based on gene expression profiling and genomic sequencing have revealed an underlying complexity and molecular heterogeneity within the disease that continues to challenge therapeutic interventions. To successfully identify and translate new treatment regimens to the clinic, it is imperative that our preclinical models recapitulate this complexity and heterogeneity. In this review article, we discuss the recent advances in development and classification of patient-derived human breast tumor xenograft models that have the potential to facilitate the next phase of drug discovery for personalized cancer therapy based on the unique driver signaling pathways in breast tumor subtypes.