Parental Xenografts Research Articles

Microvascularized tumor assembloids model for drug delivery evaluation in colorectal cancer-derived peritoneal metastasis.

Peritoneal metastasis (PM) is a fatal state of colorectal cancer, and only a few patients may benefit from systemic chemotherapy. Although hyperthermic intraperitoneal chemotherapy (HIPEC) brings hope for affected patients, the drug development and preclinical evaluation of HIPEC are seriously lagging behind, mainly due to the lack of an ideal in vitro PM model that makes drug development over-reliant on expensive and inefficient animal experiments. This study developed an in vitro colorectal cancer PM model [microvascularized tumor assembloids (vTA)] based on an assembly strategy of endothelialized microvessels and tumor spheroids. Our data showed that the in vitro perfusion cultured vTA could maintain a similar gene expression pattern to their parental xenografts. Also, the drug penetration pattern of the in vitro HIPEC in vTA could mimic the drug delivery behavior in tumor nodules during in vivo HIPEC. More importantly, we further confirmed the feasibility of constructing a tumor burden-controlled PM animal model using vTA. In conclusion, we propose a simple and effective strategy to construct physiologically simulated PM models in vitro, thus providing a basis for PM-related drug development and preclinical evaluation of locoregional therapies. STATEMENT OF SIGNIFICANCE: This study developed an in vitro colorectal cancer peritoneal metastasis (PM) model based on microvascularized tumor assembloids (vTA) for drug evaluation. With perfusion culture, vTA could maintain a similar gene expression pattern and tumor heterogeneity to their parental xenografts. And the drug penetration pattern in vTA was similar to the drug delivery behavior in tumor nodules under in vivo treatment. Moreover, vTA was more conducive to construct PM animal models with controllable tumor burden. In conclusion, the construction of vTA could provide a new strategy for the PM-related drug development and preclinical evaluation of locoregional therapies.

Abstract P4-02-01: Targeting insulin receptor in estrogen receptor positive breast cancer

Abstract Obesity and metabolic dysfunction are on the rise in the United States. Hyperinsulinemia, frequently seen in these conditions, correlates with an increased risk of development and mortality from estrogen receptor (ER) positive breast cancer. Expression of components of the insulin and insulin-like growth factor (IGF) family of proteins promote cancer cell growth in vitro, link to shorter progression-free survival, and is associates with an increased risk of metastatic disease and death in breast cancer patients. Previously our lab found that development of resistance to the commonly used ER-targeting drug tamoxifen (Tam) in vitro is associated with a loss of the type I IGF receptor expression with an increased dependence on the homologous insulin receptor (IR). These data argue for targeting IR in breast cancer, but concern has been raised due to the potential of disrupting glucose homeostasis in patients with or without existing metabolic disease. To address this concern, a novel insulin-Fc fusion protein (AKS-130) was developed to lower blood glucose levels without causing clinical hypoglycemia with a potential to target tumor IR. AKS-130 was found to downregulate IR expression in models of colorectal cancer (HCT-116) and malignant melanoma (WM266.4). Further, AKS-130 suppressed xenograft growth in fed and fasted states. We examined if AKS-130 had similar effects on ER positive breast cancer cells. To test the hypothesis that resistance to endocrine therapy increases the reliance of ER positive breast cancer cells on IR signaling, we utilized the ER positive breast cancer cell lines (MCF-7L, T47D) and their Tam resistant derivatives (MCF-7L TamR, T47D TamR). Similar to in HCT-116 and WM266.4 cells, we found that AKS-130 is a modest agonist of IR in ER+ breast cancer cells. Exposure to AKS-130 led to a marked reduction in IR expression in both parental and TamR cell lines, and was associated with partially suppressed downstream signaling to Akt. Interestingly, AKS130 induced IR downregulation was inhibited by pretreatment of BMS 536924, a dual IGF1R/IR kinase inhibitor. In vivo, AKS-130 treatment by itself scaled down MCF7L parental xenograft growth as compared to vehicle group, and AKS-130 in combination with Tam trended toward extending time to a tumor size of 500mm3. In MCF7L TamR xenografts, addition of AKS-130 to Tam treatment also trended toward delaying xenograft growth, though it was not statistically significant. Importantly, prolonged treatment with AKS-130 did not lead to significant changes in weight or blood glucose. Taken together these data show that novel agents, such as AKS-130, may serve as way to disrupt IR signaling in cancer cells without affecting host glucose metabolism. Addition of AKS-130 to Tam treatment trended toward delaying xenograft growth though further studies are needed, especially animal model systems of insulin resistance. These data indicate that IR signaling may represent a target to overcome or delay endocrine therapy resistance in breast cancer. Citation Format: Jingran Cao, Lynsey M. Fettig, Kelly LaPara, Xihong Zhang, Sylaja Murikipudi, Andrea R. Delpero, Thomas M. Lancaster, Todd C. Zion, Douglas Yee. Targeting insulin receptor in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-01.

Abstract 1670: Characterization of the chick chorioallantoic membrane tumor model in comparison with various xenograft mouse tumors

Abstract Background: The chick chorioallantoic membrane (CAM) tumor model is an in vivo three-dimensional culture model that is easy to use and inexpensive, has no ethical issues, and has been used in cancer research, although the success rate of generating CAM tumors varies among reports. We analyzed the success rate of CAM tumors from patient-derived xenograft (PDX) of esophageal squamous cell carcinoma (ESCC) and many cell line-derived xenograft (CDX) tumors of different cancer types, and we sought to determine which factors affected the success rate of generating CAM tumors. Method: We used patient cancer tissue (ESCC) and/or commercially available 12 tumorigenic cancer cell lines (esophageal cancer [TE-11 and HCE4], pancreatic cancer [CFPA-1, MIA PaCa2, and PANC-1], lung cancer [A549 and H358], skin cancer [A431 and B16F10], and biliary tract cancer [HuCCT-1, TFK-1, and MzchA2]) to generate xenograft tumors. Small (1- to 2-mm) pieces of xenograft tumors were grafted onto the CAMs of fertilized eggs. After incubating for an additional 7 to 9 days at 37.5°C and 65% humidity, nodules were observed on the assigned locations of each CAM. On the basis of hematoxylin and eosin (H&E) staining, we calculated the success rate of the CAM tumor engraftment. H&E staining was also performed with parental PDX or CDX tumors as well as parental ESCC tissues, to compare them with CAM tumors. Results: A total of 29 xenograft tumors were transplanted onto CAMs. The overall success rate in generating CAM tumors was 19/29 (66%). The CAM tumors were histologically quite similar to those of their parental CDX or PDX tumors, and the CAM tumors from PDX ESCC were also histologically similar to their parent ESCC tumors. Tumor pieces from poorly-differentiated xenograft tumors (including PDX ESCC) generated CAM tumors with a 90% success rate (19/21), in comparison with well-differentiated xenograft tumors, whose success rate was 0% (0/8). Conclusion: The overall success rate in generating CAM tumors, especially from poorly differentiated tumors, seems acceptable. The histological similarity between CAM tumors and both parent xenograft and parent tumors from humans indicates that the CAM tumor model is a promising in vivo model for the study of poorly differentiated tumors. As for well-differentiated tumors, this model has to be further optimized to increase the success rate of engraftment. Citation Format: Tomoki Saito, Shinya Ohashi, Ayaka Mizumoto, Osamu Kikuchi, Kotaro Matsumoto, Aoi Komatsu, Seiji Naganuma, Yoshihiro Yamamoto, Kenshiro Hirohashi, Masahiro Yoshioka, Masashi Tamaoki, Makiko Funakoshi, Fuyuhiko Tamanoi, Manabu Muto. Characterization of the chick chorioallantoic membrane tumor model in comparison with various xenograft mouse tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1670.

Establishment and characterisation of a new patient-derived model of myxoid liposarcoma with acquired resistance to trabectedin

BackgroundMyxoid liposarcoma is a histological subtype of liposarcoma particularly sensitive to trabectedin. In clinical use this drug does not cause cumulative toxicity, allowing prolonged treatment, generally until disease progression. No other effective therapies are available for trabectedin-resistant patients.MethodsThrough repeated in vivo treatment in athymic nude mice, we have obtained a patient-derived xenograft with acquired resistance to trabectedin.ResultsAt basal level, the morphology of the resistant and sensitive models did not differ, in keeping with the finding that the transcriptional profiles of the resistant and sensitive tumours were very similar. After trabectedin treatment adipogenesis was induced in the parental xenograft but not in the resistant one, as assessed by pathological and molecular analysis. A defective transcription-coupled-nucleotide excision repair in the resistant tumour due to mutation of the UVSSA gene may be implicated in the mechanism of resistance.ConclusionsThis is the first in vivo model of myxoid liposarcoma with acquired resistance to trabectedin. Although further studies are necessary to characterise the resistance mechanisms, this is a useful tool for studying new therapeutic strategies to overcome trabectedin resistance in patients.

Abstract 5285: The role of HIV-1 TAT interactive protein 2 in modulating tumor response to hypoxia

Abstract While there is ample evidence that HIV-1 TAT interactive protein 2 (HTATIP2) is a tumor suppressor exerting its pro-apoptotic and anti-metastatic activities by regulating the expression of a subset of pro-apoptotic and anti-apoptotic genes and metastasis suppressor genes, it remains unclear if HTATIP2 has a role in tumor response to hypoxia. The objective of this study was to define the role of HTATIP2 in mediating certain molecular and cellular changes in response to hypoxia in tumors. We employed a series of isogenic A549 human lung adenocarcinoma sublines that were genetically deficient for HTATIP2 and/or HIF-1α to examine the potential modulatory effect of HTATIP2 on oncogenic signaling pathways mediated by HIF-1α and HIF-2α, and established the HTATIP2-deficient A549 xenograft model to explore the impact of deletion of HTATIP2 on tumor response to hypoxia. Results from the human HIF-regulated cDNA plate array study revealed that under hypoxic condition (0.5% O2), several genes promoting tumor metastasis and resistance to apoptosis and anticancer treatment, including HIF1α, CYSD, MMP1, MXI1, NDRG2, PAI1 and NDRG1, were upregulated to a greater extent in HTATIP2 knockdown A549 cells than in the control A549 cells. In contrast, genes involved in angiogenesis, glucose metabolism and protection of apoptosis under hypoxia, such as PDGF, PDK1, CA9, and SAG, were upregulated to a greater extent in control A549 cells than in HTATIP2-knockdown A549 cells. Results from the in vivo study demonstrated that the mean growth rate of HTATIP2-deficient A549 xenograft tumors was significantly increased as compared with that of parental A549 xenograft tumors irrespective of the presence or absence of sorafenib treatment. Moreover, the data of photoacoustic imaging (PAI) of tumor hypoxia revealed that the parental A549 tumors were better oxygenated than the counterpart HTATIP2-deficient A549 tumors. After the 10-day sorafenib treatment, the oxygenated regions in sorafenib-treated parental A549 tumors were significantly reduced as compared with the vehicle-treated parental A549 tumors, whereas no significant change in the oxygenated regions was found between vehicle- and sorafenib-treated HTATIP2-deficient A549 tumors. The overall finding of our study suggests that downregulation of HTATIP2 affords growth advantage to tumor cells by fine-tuning tumor adaptation to hypoxia, leading to tumor progression and aggression. Citation Format: Qingyu Stephanie Zhou, Xiaofang Guo, Minghua Li, Hua Pan. The role of HIV-1 TAT interactive protein 2 in modulating tumor response to hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5285.

Abstract 3113: Regulation of TOR complex 1 by the BRAFV600EMutant determines response and development of resistance to MEK inhibition by trametinib in glioma models

Abstract Pediatric Low-Grade glioma (PLGG) is the most common brain tumor in children. Current treatment protocols, particularly those involving high dose radiation therapy are associated with neurocognitive and neuroendocrine dysfunction. Genomic studies have revealed that PLGG are characterized by activation of the MAPK cascade. Recently, the value of selumetinib, an allosteric inhibitor of MEK1/2 has been demonstrated in PLGG. However, drug resistance will inevitably limit the efficacy. Trametinib is a more potent MEK1/2 inhibitor with better brain penetration than selumetinib. Here we have attempted to identify the mechanism/s of the resistance to trametinib that PLGG developes, and to explore combination treatments that may retard or prevent emergence of trametinib resistance in animal models. In BT-40 (childhood anaplastic astrocytoma with BRAFV600E mutation) xenografts, trametinib caused complete tumor regressions, followed by tumor regrowth at the end of treatment. Trametinib treatment rapidly induced pSTAT3(Y705), that was blocked by the JAK1/2 inhibitor, ruxolitinib. Agaist BT-40 xenografts, ruxolitinib slightly enhanced the antitumor response to trametinib. The combination of trametinib with rapamycin was more active in each xenograft line than either single agent alone and in both subcutaneous and intracranial BT-40 models. In BT-40 xenografts, resistance to trametinib was consistently induced within 3 or 4 6-week cycles of treatment similar to that for the trametinib + ruxolitinib combination. In contrast, resistance to combined trametinib and rapamycin was not developed. Rapamycin prevented emergence of trametinib resistance, but did not reverse resistance to trametinib, once acquired. Inhibition of MAPK signaling by trametinib was similar in parental and trametinib-resistant BT-40 xenografts, however, trametinib reistant tumor cells failed to undergo apoptosis. Cells freshly isolated from trametinib resistant BT-40 xenografts had a higher apoptotic threshhold, whereas cells isolated from BT-40 xenografts after 3 cycles of trametinib + rapamycin, remained equally sensitive to parental BT-40 cells, as determined by BH3 profiling. This study reveals a potential new therapeutic use of rapamycin, preventing emergence of resistance to trametinib. Potentially, addition of rapamycin to MEK inhibitor therapy children with BRAF-driven low-grade glioma may be of value. Citation Format: Fuyang Li. Regulation of TOR complex 1 by the BRAFV600EMutant determines response and development of resistance to MEK inhibition by trametinib in glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3113.

Development of a Cross-Reactive Monoclonal Antibody for Detecting the Tumor Stroma.

Here, we document the discovery of a monoclonal antibody that selectively binds to both human and murine fibroblast activation protein alpha (FAP), a serine protease that is overexpressed on cancer-associated fibroblasts (CAFs), making it an attractive therapeutic target for the aiding and abetting tumor microenvironment. The lead antibody, B12, was identified from a naïve murine single-chain variable fragment antibody phage display library screened against recombinant human FAP on magnetic beads. The heavy and light chains of B12 were cloned into full-length human immunoglobulin 1 (IgG) vectors and expressed as a chimeric monoclonal antibody (B12 IgG). We engineered a drug-resistant prostate cancer cell line, CWR-R1-EnzR, to express human FAP for antibody characterization and validation (R1-EnzRFAP). B12 IgG selectively bound to the R1-EnzRFAP cells by flow cytometry and was internalized in vitro by confocal microscopy. B12 IgG was further evaluated as a near-infrared (NIR) optical imaging probe in R1-EnzRFAP and parental xenograft models. High tumor uptake and retention of the NIR probe was observed in the R1-EnzRFAP xenografts, and endogenous expression of murine stromal origin FAP was detected in the parental xenografts. Ex vivo evaluation of these models by immunohistochemistry documented B12 IgG localization to both human and murine FAP-expressing cells.

Abstract 519: Antitumor activity of MET antibody emibetuzumab (LY2875358) in combination with EGFR inhibitors in erlotinib resistant (ER) xenograft mouse models

Abstract Background: MET amplification (amp) is a resistance mechanism to EGFR TKI treatment. Emibetuzumab, a bivalent MET antibody (Ab) blocks HGF binding to MET and internalizes the receptor. Combination of emibetuzumab with EGFR TKIs (erlotinib, AZD9291, CO1686) or EGFR Ab (necitumumab, cetuximab) was evaluated in 3 ER xenograft models. Methods: Model 1: ER cell line HCC827ERL with high focal MET amp, high pMET, EGFR ex19 del (no T790M) was created from parental HCC827 NSCLC (EGFR ex19 del, EGFR amp, no MET amp) by increasing concentration of erlotinib in vitro over 7 months. Model 2: ER cell line HCC827-A8 was derived from HCC827 parental xenograft tumor serially passed in vivo with long term treatment of gefitinib and erlotinib. HCC827-A8 cells express high focal MET amp, high pMET/AXL (Western blot) while retaining EGFR ex19 del (no T790M). Model 3: LU0858 was an ER patient-derived NSCLC xenograft tumor, with focal MET amp, EGFR L858R (no T790M). MET amp and EGFRmt was determined by FISH and LNA-PCR sequencing respectively. Compound dosing: emibetuzumab 20 mg/kg qw; necitumumab 4 mg/kg or 20 mg/kg biw; cetuximab 4 mg/kg biw; erlotinib 25 mg/kg qd; 5 mg/kg AZD9291 qd; 30 mg/kg CO1686 bid. Results: EGFR inhibitors, but not emibetuzumab showed significant single agent anti-tumor effect in xenograft tumors derived from non-MET amp HCC827 parental cells. In MET amp ER models, single agent emibetuzumab resulted in tumor growth inhibition in Model 1 (T/C= 51.7%-61.0%, p<0.05) and 3 (T/C=2.8%, p<0.05)] but no tumor regression, and no anti-tumor effect in Model 2. Where evaluated, EGFR inhibitors showed no anti-tumor effect in the 3 ER models as monotherapy, except necitumumab (20 mg/kg) in Model 1 (T/C = 36.2%, p<0.05). However, combination of emibetuzumab with AZD9291, CO1686, necitumumab (20 mg/kg), or erlotinib resulted in 80.4%, 58.2%, 44.4%, 69.1% tumor regression respectively (p<0.001) in Model 1, while emibetuzumab + cetuximab (4 mg/kg) resulted in tumor stasis (T/C=0.2%, p<0.05). In Model 2, emibetuzumab + AZD9291 resulted in tumor stasis (T/C = 12.9%, p<0.05). In Model 3, emibetuzumab + necitumumab (20 mg/kg) resulted in 80.1% tumor regression (p<0.001). Conclusion: The three erlotinib resistant models with MET amp and retaining sensitizing EGFRmt (ex19 del or L858R), and no acquired T790M were found resistant to other EGFR inhibitors (Abs and TKIs). Emibetuzumab in combination with either EGFR TKI or Ab showed anti-tumor activity in MET amp ER xenograft models including tumor regression in 2 out of 3 models. The combination of emibetuzumab with erlotinib is being evaluated in NSCLC patients with EGFR activating mutation (NCT01897480). Citation Format: Suzane L. Um, Victoria L. Peek, Jennifer R. Stephens, Jessica A. Baker, Holly K. Cannon, Joel D. Cook, Isabella H. Wulur, Roger Agyei, Sudhakar Chintharlapalli, Robert J. Evans, William J. Feaver, Lysiane Huber, Linda N. Lee, Ling Liu, Liandong Ma, Ruslan Novosiadly, Volker Wacheck, Sau-Chi Betty Yan. Antitumor activity of MET antibody emibetuzumab (LY2875358) in combination with EGFR inhibitors in erlotinib resistant (ER) xenograft mouse models [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 519. doi:10.1158/1538-7445.AM2017-519

Abstract P3-04-10: Utility of the orally bioavailable selective estrogen receptor degrader AZD9496 in ESR1 mutant preclinical models of estrogen receptor positive breast cancer

Abstract Approximately 70% of breast cancers express estrogen receptor alpha (ERα) and anti-hormonal therapies which either block the production of estrogen (e.g. anastrozole) or directly block ERα function (e.g. tamoxifen) remain the mainstay of treatment for these patients. While these therapies are highly effective resistance can occur. A common resistance mechanism to anti-hormonal agents is the mutation of ESR1, the gene that encodes ERα, which leads to ligand independent activity. Evidence is emerging that the selective estrogen receptor degrader (SERD) fulvestrant is effective in patients with ESR1 mutations. However, given the low bioavailability of fulvestrant and the still detectable levels of ER in clinical samples after treatment with fulvestrant it is hypothesised that SERDs with improved pharmacokinetic properties which are able to drive greater degradation of ERα may provide additional clinical benefit. We have previously described the discovery and characterisation of the orally bioavailable SERD AZD9496, which is currently in phase I clinical trials in ER+ breast cancer patients. Here we report the preclinical activity of AZD9496 in cell line and patient derived xenograft models expressing clinically relevant ESR1 mutations. We have engineered MCF-7 cells to express Y537S ESR1 which confers the ability to proliferate in the absence of estradiol, consistent with ligand independent activation of ER signalling. AZD9496 is able to inhibit proliferation of these cells and downregulate progesterone receptor protein expression at low nanomolar concentrations. Furthermore, when MCF-7 Y537S ESR1 cells are implanted as xenografts they grow in the absence of exogenous estradiol, are as sensitive to AZD9496 as parental MCF7 xenografts and demonstrate downregulation of ER dependent biomarkers. AZD9496 also has anti-tumour and pharmacodynamic efficacy in patient derived xenograft models expressing D538G ESR1. Taken together, these data strengthen the body of data suggesting that SERDs may be active in patients with tumours containing ESR1 mutations and supports the inclusion of this patient population in AZD9496 clinical trials. Citation Format: Morrow CJ, Lawson M, Ladd B, de Almeida C, James N, Curwen JO, Hanson L, Bell K, Goeppert A, Fisher D, Gangl E, De Savi C, Scott JS, D'Cruz C, Klinowska T, Weir HM. Utility of the orally bioavailable selective estrogen receptor degrader AZD9496 in ESR1 mutant preclinical models of estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-10.

Mitochondrial deficiency impairs hypoxic induction of HIF-1 transcriptional activity and retards tumor growth.

Mitochondria can be involved in regulating cellular stress response to hypoxia and tumor growth, but little is known about that mechanistic relationship. Here, we show that mitochondrial deficiency severely retards tumor xenograft growth with impairing hypoxic induction of HIF-1 transcriptional activity. Using mtDNA-deficient ρ0 cells, we found that HIF-1 pathway activation was comparable in slow-growing ρ0 xenografts and rapid-growing parental xenografts. Interestingly, we found that ex vivo ρ0 cells derived from ρ0 xenografts exhibited slightly increased HIF-1α expression and modest HIF-1 pathway activation regardless of oxygen concentration. Surprisingly, ρ0 cells, as well as parental cells treated with oxidative phosphorylation inhibitors, were unable to boost HIF-1 transcriptional activity during hypoxia, although HIF-1α protein levels were ordinarily increased in these cells under hypoxic conditions. These findings indicate that mitochondrial deficiency causes loss of hypoxia-induced HIF-1 transcriptional activity and thereby might lead to a constitutive HIF-1 pathway activation as a cellular adaptation mechanism in tumor microenvironment.

Abstract 283: PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances pemetrexed antitumor efficacy in a human nonsquamous NSCLC xenograft model

Abstract Introduction: Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that has been shown to accumulate at high levels in several cancers, including non-small cell lung cancer (NSCLC). The accumulation of HA in NSCLC is associated with a more aggressive phenotype and shortened overall survival. In a prevalence analysis using tissue microarrays of archival human specimens (N = 194) stained by affinity histochemistry, we demonstrated that 55% of adenocarcinomas, 74% of squamous cell carcinomas and 83% of large cell carcinomas accumulated HA. The engineered enzyme PEGylated recombinant human hyaluronidase PH20 (PEGPH20) removes HA from the tumor microenvironment. PEGPH20-based therapies are being evaluated in clinical studies of several solid tumors, including NSCLC (NCT02346370, NCT02563548). Herein, we evaluated the hypothesis that PEGPH20 combination therapy could enhance the efficacy of pemetrexed (PEM), a folate antimetabolite approved by FDA as a first-line treatment in combination with cisplatin, against locally advanced and metastatic NSCLC in patients with non-squamous histology. Methods: We evaluated PEGPH20 plus PEM in a NSCLC xenograft model. First, the human NSCLC adenocarinoma cell line A549 was transduced with hyaluronan synthase-3 (HAS3) to generate the A549/HAS3 cell line. Next, A549/HAS3 cells were inoculated adjacent to the tibial periosteum of nude mice and tumor growth was monitored via ultrasonography. When tumors size reached ∼250 mm3, mice were staged into six groups: (1) vehicle; (2) PEGPH20, (3) PEM daily, (4) PEM weekly, (5) PEGPH20+PEM daily or (6) PEGPH20+PEM weekly. Vascular volume, hypoxia and tumor growth inhibition (TGI) were assessed using conventional methods. Affinity histochemical staining for HA was performed on formalin-fixed paraffin-embedded tissue sections after harvest. Results: A549/HAS3 tumors grew faster than parental A549 tumors in vivo. PEGPH20 monotherapy significantly reduced tumoral HA, increased vascular volume and reduced hypoxia in tumors. Further, TGI with PEGPH20+PEM, weekly or daily, was 29.7 or 31.1%, respectively, whereas in either PEGPH20 alone or PEM alone, both were <9%. Staining of A549/HAS3 tumors showed increased accumulation of HA as compared with the parental A549 xenografts. Conclusion: These data suggest that addition of PEGPH20 to pemetrexed increases the efficacy of PEM and support further investigation of PEGPH20 plus PEM treatment in NSCLC tumors of non-squamous histology that accumulate HA. Citation Format: Renee Clift, Jessica A. Cowell, Susan J. Zimmerman, Mathieu Marella, Ping Jiang, Arnold B. Gelb, Michael J. LaBarre, Daniel C. Maneval, Curtis B. Thompson, Xiaoming Li. PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances pemetrexed antitumor efficacy in a human nonsquamous NSCLC xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 283.

Abstract 2669: Glutaminase alternative splicing controls tumor growth and metabolism

Abstract Cancer cells reprogram their metabolism to meet their energetic and biosynthetic needs, with many cancer cells showing increased aerobic glycolysis and glutamine consumption. In addition to providing carbon to the TCA cycle to compensate for the diversion of glucose to lactate, glutamine is an important carbon and nitrogen donor for the synthesis of nucleotides, amino sugars and non-essential amino acids. Glutaminase (GLS) catalyzes the first step of glutamine metabolism, the hydrolysis of glutamine to glutamate, which can then be converted to alpha-ketoglutarate, glutathione or non-essential amino acids. GLS is alternatively spliced to make the KGA or more catalytically active GAC isoforms. Analysis of publicly available TCGA RNA-seq data from primary human tumors shows that GAC is the predominant splice form of GLS in many tumor types. However, the roles played by GAC versus KGA in tumor metabolism and progression are poorly understood. To facilitate the study of GLS alternative splicing, CRISPR/Cas9 mediated deletion of the GAC specific exon was used to convert predominantly GAC-expressing parental cell lines to solely KGA-expressing cell lines (ΔGAC). Despite showing decreased glutamine-derived glutamate and α-ketoglutarate, ΔGAC cells do not show impaired growth in nutrient-replete in vitro conditions compared to parental cells. 13C-isotopic labeling and metabolomics showed that ΔGAC cells have decreased levels of proline and alanine, both of which can be derived from glutamine-derived glutamate. This led us to ask if ΔGAC cells are dependent on exogenous non-essential amino acids. We found that depletion of non-essential amino acids slowed the growth of ΔGAC cells compared with parental cells, rescuable by exogenous proline and alanine. Importantly, the growth of lung (A549) and breast cancer (MDA-MB231) ΔGAC xenograft models was diminished compared with parental tumor xenografts in vivo, which is consistent with a role for GAC in nutrient challenged conditions. In summary, the GAC form of glutaminase fuels tumor growth partly through synthesis of non-essential amino acids, particularly under nutrient limiting conditions. As GLS inhibitors enter the clinic, a more thorough understanding of the role of GLS in tumor metabolism will aid in the development of treatment strategies to best make use of these drugs. Citation Format: Zachary Stine, Zandra E. Walton, Lin Zhang, Teresa MW Fan, Andrew N. Lane, Chi V. Dang. Glutaminase alternative splicing controls tumor growth and metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2669.

Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture

Commercially available, highly passaged pancreatic cancer (PC) cell lines are of limited translational value. Attempts to overcome this limitation have primarily consisted of cancer cell isolation and culture directly from human PC specimens. However, these techniques are associated with exceedingly low success rates. Here, we demonstrate a highly reproducible culture of primary PC cell lines (PPCLs) from patient-derived xenografts, which preserve, in part, the intratumoral heterogeneity known to exist in PC. PPCL expansion from patient-derived xenografts was successful in 100% of attempts (5 of 5). Phenotypic analysis was evaluated with flow cytometry, immunofluorescence microscopy, and short tandem repeat profiling. Importantly, tumorigenicity of PPCLs expanded from patient-derived xenografts was assessed by subcutaneous injection into nonobese diabeteic.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice. Morphologically, subcutaneous injection of all PPCLs into mice yielded tumors with similar characteristics to the parent xenograft. PPCLs uniformly expressed class I human leukocyte antigen, epithelial cell adhesion molecule, and cytokeratin-19. Heterogeneity within each PPCL persisted in culture for the frequency of cells expressing the cancer stem cell markers CD44, CD133, and c-Met and the immunologic markers human leukocyte antigen class II and programmed death ligand 1. This work therefore presents a reliable method for the rapid expansion of primary human PC cells and, thereby, provides a platform for translational investigation and, importantly, potential personalized therapeutic approaches.

Abstract B31: Characterization of a novel model of prostate cancer metastasis resulting from androgen receptor suppression

Abstract Background: Prostate cancer (PC) is the second leading cause of male cancer-related mortality: 30,000 men die each year from metastatic disease. The Androgen Receptor (AR) may be the earliest known example of a lineage oncogene – a master regulator of prostate cancer cell survival and growth. The AR protein is detectable at all stages of prostate cancer progression and is the primary target of pharmacological agents capable of suppressing AR signaling to extend survival in men with metastatic prostate cancer (mPC). The CYP17 inhibitor Abiraterone, and the 3rd generation AR antagonist, MDV3100 prolong the survival of men with mPC, though cures remain exceeding rare using these agents individually. In order to develop additional therapies to be used in combination with current AR inhibitors, preclinical models that mimic PC progression need to be established. Tissue microarray studies from hormone-naïve, nodal positive, prostate cancer patients revealed a significant down-regulation of AR protein level in matched nodal metastases versus primary tumors. These results suggest that loss of AR or AR activity could play a role in the progression to metastatic disease. Methods: We used a short-hairpin RNA targeting exon 1 of the AR to reduce AR protein in vivo using a doxycycline-inducible system in the non-metastatic AR-positive human prostate cancer cell line 22Rv1. This cell line model, derived from a primary tumor, is castration-resistant and enzalutamide/MDV3100-resistant. We generated xenografts using 22Rv1shARex1 and evaluated tumor growth and propensity for metastatic spread. We profiled transcriptional changes in the context of AR suppression and compared gene expression changes between grafted tumors and sites of metastatic spread. Results: Xenograft studies in NOD-SCID mice revealed that reduction of the AR protein decreased tumor proliferation rates and promoted cancer metastasis. Tumor-bearing mice exposed to continual reduction of the AR developed large liver metastases and had significantly increased disseminated tumor cells in their bone marrow. Furthermore, intratibial and intracardiac injection of 22Rv1shARex1 followed by AR reduction, resulted in an increased formation of metastases. Transcriptional analysis revealed unique signatures between untreated parental 22Rv1 xenografts, tumors with AR reduction, and liver metastases. Genes up-regulated in xenografts following AR reduction, were also up-regulated in patient metastases versus primary tumors including transcripts encoding EGF, HGF, and ZEB2. Discussion: The AR is the primary therapeutic target for mPC. However, a cure for advanced cancer and metastatic disease remains elusive. Reduction of the AR in the 22Rv1 in vivo model mimics disease progression seen in patients. Molecular changes observed during the transformation of localized cancer to metastatic disease in this model may identify new targets to be used in combination with current therapies intent on ablating AR signaling. Citation Format: Clinton K. Matson, Peter S. Nelson. Characterization of a novel model of prostate cancer metastasis resulting from androgen receptor suppression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B31.

Abstract 765: Overcoming acquired EGFRi resistance in NSCLC with targeted beam irradiation in combination with targeted agents

Abstract BACKGROUND: Non-small cell lung cancer (NSCLC) patients undergo primary, adjuvant or neoadjuvant radiotherapy treatment with image-guided radiotherapy (IGRT) being widely used to provide more accurate treatment plans and reduced side effects. Patients who have activating mutations in the epidermal growth factor receptor (EGFR) gene are treated with EGFR tyrosine kinase inhibitors (EGFRi). However, resistance to EGFRi occurs in the majority of patients with the main clinical drivers being secondary gatekeeper mutations (T790M) or amplification of genes such as c-MET and Her2. The small animal radiation research platform (SARRP), which allows the treatment of rodent models of cancer more accurately with planned protocols similar to those utilised in the clinic, was used in combination with EGFRi, to assess the efficacy of combination treatment strategies of irradiation (IR) in EGFRi resistant NSCLC xenografts. METHODS: Resistance to EGFRi in the NSCLC cell line HCC827, which harbours an activating EGFR mutation, was generated through repeated exposure to Gefitinib or Erlotinib. Resistant models were characterised for further mutations in the EGFR gene by direct sequencing and for c-MET, Axl and Her2 over-expression/genomic amplification by quantitative PCR. EGFRi resistant or parental HCC827 cells were implanted subcutaneously into nude mice, xenograft growth was monitored by calliper measurements and treatment initiated when the mean tumour volume reached ∼200mm3. An image guided IR dose of 3Gy/mouse/day (30Gy total) was targeted directly to the xenografts whilst sparing the surrounding normal tissue. Mice were treated with IR either alone or in combination with Erlotinib or Crizotinib; response to treatment was evaluated by tumour volume measurement. RESULTS: EGFR-TKI resistant HCC827 cell lines were generated following successive cycles of treatment. Amplification of c-MET was identified in a number of clones, but no Her2 amplification or gatekeeper mutations; therefore Crizotinib, a c-MET inhibitor, was selected for evaluation in these models. When assessed in vivo, treatment of parental HCC827 xenografts with targeted IR or Erlotinib treatment resulted in tumour regression. However, in the c-MET driven resistant model, both the IR and Erlotinib response was significantly attenuated, resulting in tumour re-growth. Combination testing with Crizotinib restored the efficacy of both IR and Erlotinib to naïve treatment levels confirming the role of c-MET in resistance. CONCLUSIONS: Resistance to EGFRi and IR in a HCC827 model is driven by c-MET amplification. Treatment with Crizotinib restored sensitivity to both EGFRi and IR demonstrating that pre-clinical models of resistance are invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent/overcome resistance to EGFRi. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Lucy Page, Simon Jiang, Jane Wrigley, Yinfei Yin, Qian Shi, Martin Page, Ian Wilson, Rajendra Kumari. Overcoming acquired EGFRi resistance in NSCLC with targeted beam irradiation in combination with targeted agents. [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 765. doi:10.1158/1538-7445.AM2015-765

Radiation therapy may increase metastatic potential in alveolar rhabdomyosarcoma.

We previously determined that radiation could be safely administered using a mouse-flank in vivo model to both alveolar (Rh30) and embryonal (Rh18) rhabdomyosarcoma xenografts. Mice from both tumor lines in this experiment developed metastases, an event not previously described with these models. We sought to determine if radiation-induced changes in gene expression underlie an increase in the metastatic behavior of these tumor models. Parental Rh18 and Rh30 xenografts, as well as tumor that recurred locally after radiotherapy (Rh18RT and Rh30RT), were grown subcutaneously in the flanks of SCID mice and then subjected to either fractionated radiotherapy or survival surgery alone. Metastasis formation was monitored and recorded. Gene expression profiling was also performed on RNA extracted from parental, recurrent, and metastatic tissue of both tumor lines. Rh30 and Rh30RT xenografts demonstrated metastases only if they were exposed to fractionated radiotherapy, whereas Rh18 and Rh18RT xenografts experienced significantly fewer metastatic events when treated with fractionated radiotherapy compared to survival surgery alone. Mean time to metastasis formation was 40 days in the recurrent tumors and 73 days in the parental xenografts. Gene expression profiling noted clustering of Rh30 recurrent and metastatic tissue that was independent of the parental Rh30 tissue. Rh18RT xenografts lost radiosensitivity compared to parental Rh18. Radiation therapy can significantly decrease the formation of metastases in radio-sensitive tumors (Rh18) and may induce a more pro-metastatic phenotype in radio-resistant lines (Rh30).

88 Genome-wide drug sensitivity screens in haploid mouse embryonic stem cells

osteosarcoma cell line in vitro and in vivo. The SJSA-1 cell line possesses an amplified MDM2 gene and wild-type p53 and is sensitive to SAR405838 and other MDM2 inhibitors in vitro and in vivo. In vitro treatment of the SJSA-1 cells with SAR405838 using two different treatment protocols resulted in acquired resistance to the drug. Analysis of these resistant cells showed that p53 is mutated in the DNA binding domain and cannot be activated by SAR405838. Treatment of mice bearing the parental SJSA-1 xenograft tumors with SAR405838 led to rapid tumor regression, but tumors eventually returned after treatment cessation. A number of cell lines were established by culturing the regrown tumors and, surprisingly, these sublines showed minimal loss of sensitivity to SAR405838 as compared to the parental SJSA-1 cell line. Consistently, analysis of thesesublines showed that p53 retains its wild-type status, with the exception of one subline, that harbored a single heterozygous mutation C176F. Computational modeling suggested that the p53 C176F mutant may still be capable of binding to target DNA to elicit gene transcription, which was confirmed by in vitro treatment with SAR405838. Xenografts of representative sublines lacking p53 C176F mutation obtained from regrown tumors treated with one-round SAR405838 were still responsive to SAR405838 treatment. Interestingly, after a second round of in vivo treatment with SAR405838, all sublines established from harvested tumors uniformly harbored a heterozygous C176F p53 mutation, which were still responsive to SAR405838, albeit with reduced sensitivity. Our study showed that the SJSA-1 tumor cells acquired profound resistance to MDM2 inhibitor SAR405838 in vitro and developed inactivating p53 mutations in its DNA binding domain. But in vivo, SJSA-1 tumor cells only acquired minimal or modest resistance when treated with either oneor two-rounds of SAR405838. Our study suggests that tumor cells respond differently to drug treatment under in vitro and in vivo environments and consequently develop different types of resistance.

Abstract LB-212: HER2-overexpressing breast cancer xenografts amplify FGFR signaling upon acquisition of resistance to dual blockade with trastuzumab and lapatinib

Abstract We sought to discover mechanisms of acquired resistance to the combination of the HER2 inhibitors trastuzumab and lapatinib in HER2 gene amplified BT474 xenografts. The combination of lapatinib and trastuzumab completely eliminated BT474 tumors established in nude mice after <3 weeks of treatment. However, some tumors recurred as early as 9 weeks after the combination was stopped. Three recurrent tumors did not respond to retreatment with the combination of lapatinib and trastuzumab. More than 90% of tumors derived from these recurrent tumors did not respond to lapatinib/trastuzumab or the combination of trastuzumab and pertuzumab. Both the parental drug-sensitive xenografts and the lapatinib/trastuzumab-resistant (LTR) tumors exhibited 5 to 6-fold HER2 gene amplification as measured by FISH using probes for HER2 and chromosome 17. The LTR cancers maintained levels of P-HER2, P-AKT and P-ERK in the presence of lapatinib and trastuzumab as measured by IHC of tumor sections whereas treatment of parental BT474 xenografts reduced P-HER2 levels. LTR tumors exhibited markedly reduced in situ levels of lapatinib compared to BT474 sensitive tumors (2.157 µg lapatinib/gm tissue versus 4.701 µg lapatinib/gm tissue, p=0.02) as assessed by liquid chromatography-mass spectrometry (LC-MS). Quantitative site-specific mass spectrometric analysis of tyrosine phosphorylation from tumor lysates revealed enriched P-Y877 HER2 in LTR compared to parental BT474 xenografts. We next performed next generation sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes. There were no mutations in HER2 but we observed amplification of FGF3, FGF4, and FGF19 genes in LTR xenografts. Consistent with increased autocrine activation of FGFR, we observed markedly elevated levels of P-FGFR in LTR xenografts compared to parental xenografts as assessed by IHC. We speculate that ligand-induced FGFR signaling may induce resistance to dual HER2 blockade by 1) a tumor cell autonomous mechanism resulting in transactivation of HER2 and, 2) an alteration in the tumor microenvironment that reduces lapatinib uptake and/or retention by the LTR tumors. In conclusion, we show amplification of FGFR signaling upon acquired resistance to combinations of HER2-targeted agents. Studies with FGFR tyrosine kinase inhibitors to reverse drug resistance are in progress. Citation Format: Joan T. Garrett, Jennifer Becker, Mónica Valeria Estrada, Carlos L. Arteaga. HER2-overexpressing breast cancer xenografts amplify FGFR signaling upon acquisition of resistance to dual blockade with trastuzumab and lapatinib. [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 LB-212. doi:10.1158/1538-7445.AM2014-LB-212

Abstract 2108: Estrogen non genomic signalling is activated in tamoxifen-resistant breast cancer

Abstract Introduction: Although the presence of nuclear estrogen receptor (ERα) is widely used to guide breast cancer therapy, less attention has been paid to the receptor cytoplasmic signalling. We have already shown that arginine methylation of ERα is essential for E2-non genomic pathway triggered by ERα interaction with PI3K and c-Src thereby propagating the signal to downstream transduction cascades (Le Romancer et al, Mol Cell, 2008). Recently, we have found that this pathway is operative in vivo and is activated in aggressive tumors representing a new potential target for breast cancer therapy (Poulard et al, Embo Mol Med, 2012). In addition, the cross talk between ERα and growth factor signalling has emerged as a critical factor in endocrine resistance. The aim of this study was to investigate the receptor cytoplasmic signalling in human ERα-positive breast cancer with acquired resistance to tamoxifen. Experimental procedures: The activation of cytoplasmic ER signalling pathway was investigated in tamoxifen resistant MCF-7 cells and in patient-derived xenografts (PDX) of ERα-positive breast cancer with acquired resistance in vivo to tamoxifen. In addition, tamoxifen-resistant PDX models were treated with tamoxifen, fulvestrant and everolimus, during 90 days. ERα interactions with Src and PI3K were investigated by Proximity Ligation Assay (PLA) in formalin-fixed tumors, a technology which enables to investigate protein/protein interactions in tissues. Results: Interestingly, we detected an increase of ERα/Src/PI3K expression in tamoxifen resistant versus parental MCF-7 cells. This result was confirmed in one PDX model where we found a significant increase of ERα/Src/PI3K expression in the tamoxifen-resistant tumor compared to the corresponding parental xenograft. Finally, the analysis of treated tumors revealed a significant decrease of ERα/Src/PI3K expression in the fulvestrant-treated tumors as well as in tumors treated by the everolimus and fulvestrant combination. In conclusion these results show that ERα cytoplasmic signalling is associated to acquisition of tamoxifen resistance both in cell line and patients-derived xenografts. In addition, ERα cytoplasmic signalling appears to be affected in tumors responding to alternative treatments such as fulvestrant or everolimus. Targeted therapies against ERα, Src and PI3K are in progress and could constitute a new approach to circumvent resistance to endocrine therapies. Citation Format: Coralie Poulard, Juliette Rambaud, Pascale Cohen, Antimo Migliaccio, Laura Corbo, Elisabetta Marangoni, Muriel Le Romancer. Estrogen non genomic signalling is activated in tamoxifen-resistant breast cancer. [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 2108. doi:10.1158/1538-7445.AM2014-2108

Abstract C174: Establishment of luminal breast cancer patient-derived xenografts with acquired resistance to both hormonotherapy and everolimus.

Abstract The addition of everolimus (Rad) to endocrine therapies (ET) in luminal advanced breast cancers has shown to increase overall survival in randomized studies. However drug resistance is an emerging problem and most patients eventually experience progression of disease. We aimed to establish and characterize patient-derived xenografts (PDX) of luminal breast cancers with acquired resistance in vivo to both ET and Rad. Two PDX xenografts (HBCx22 and HBCx34), established from early stage ER+ breast cancers, were treated in vivo with different ET (tamoxifen, fulvestrant, ovariectomy and letrozole) during 12 months and tumors escaping to therapies were re-engrafted and maintained under treatment. Four hormono-resistant (HR) xenografts were established, 2 resistant to Tamoxifen (HBCx22TamR and HBCx34TamR) and 2 resistant to ovariectomy (HBCx22OvaR and HBCx34OvaR). Expression of estrogen receptor (ER) was maintained, while expression of several ER-controlled genes (MYB, PS2 and PR) was decreased in HR tumors indicating modifications of ER transcriptional activity. The expression of ER co-regulators was additionally studied: GREB1 was reduced specifically in TamR, while FOXA1 were increased specifically in TamR. Expression analysis of P-AKT, P-mTOR and P-S6 showed similar levels of PI3 kinase pathway activation between parental and HR xenografts. Gene expression profiling, performed with Affymetrix Gene Expression Arrays, showed that genes differentially expressed in TamR and OvaR tumors were only partially overlapping, suggesting treatment-specific mechanisms of hormono-resistance. In addition, TamR and OvaR signatures were tumor-specific. Gene ontology enrichment and pathway analyses of gene expression datasets will be presented at the meeting. HR xenografts were then treated with Rad alone and combined to ET. In HBCx22OvaR and TamR, resistant to all ET, treatment with Rad arrested tumor growth in vivo but did not show any additive effect when combined to ET. Conversely, the HBCx34TamR model was still responding to fulvestrant, and the combination Rad+Fulvestrant resulted in tumor regressions. In the HBCx34TamR xenograft, tumor escape to Rad treatment occurred in one mouse after 8 months of continuous treatment. The tumor was re-engrafted and maintained as tumorgraft resistant to both tamoxifen and Rad (HBCx34Tam-RadR). Western blot and IHC studies showed that mTOR signaling was still inhibited in this tumor, but it was associated to MAP kinase activation and increased IGF-1R expression. In conclusion, we have developed PDX models of luminal breast cancer with cross-resistance to ET and everolimus in vivo. These models provide a valuable preclinical tool to investigate molecular mechanisms of acquired resistance and to test new targeted therapies and new combinations in patients’ tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C174. Citation Format: Rania El Botty, Paul Cottu, Franck Assayag, Pierre de la Grange, David Gentien, Aurélie Thuleau, Sophie Vacher, Sophie Chateau-Joubert, Ivan Bièche, Elisabetta Marangoni. Establishment of luminal breast cancer patient-derived xenografts with acquired resistance to both hormonotherapy and everolimus. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C174.