Tamika Mitchell

Au Nanomatryoshkas as Efficient Near-Infrared Photothermal Transducers for Cancer Treatment: Benchmarking against Nanoshells

Au nanoparticles with plasmon resonances in the near-infrared (NIR) region of the spectrum efficiently convert light into heat, a property useful for the photothermal ablation of cancerous tumors subsequent to nanoparticle uptake at the tumor site. A critical aspect of this process is nanoparticle size, which influences both tumor uptake and photothermal efficiency. Here, we report a direct comparative study of ∼90 nm diameter Au nanomatryoshkas (Au/SiO2/Au) and ∼150 nm diameter Au nanoshells for photothermal therapeutic efficacy in highly aggressive triple negative breast cancer (TNBC) tumors in mice. Au nanomatryoshkas are strong light absorbers with 77% absorption efficiency, while the nanoshells are weaker absorbers with only 15% absorption efficiency. After an intravenous injection of Au nanomatryoshkas followed by a single NIR laser dose of 2 W/cm2 for 5 min, 83% of the TNBC tumor-bearing mice appeared healthy and tumor free >60 days later, while only 33% of mice treated with nanoshells survived the same period. The smaller size and larger absorption cross section of Au nanomatryoshkas combine to make this nanoparticle more effective than Au nanoshells for photothermal cancer therapy.

Sub-100 nm gold nanomatryoshkas improve photo-thermal therapy efficacy in large and highly aggressive triple negative breast tumors

There is an unmet need for efficient near-infrared photothermal transducers for the treatment of highly aggressive cancers and large tumors where the penetration of light can be substantially reduced, and the intra-tumoral nanoparticle transport is restricted due to the presence of hypoxic or necrotic regions. We report the performance advantages obtained by sub 100nm gold nanomatryushkas, comprising concentric gold-silica-gold layers compared to conventional ~150nm silica core gold nanoshells for photothermal therapy of triple negative breast cancer. We demonstrate that a 33% reduction in silica-core-gold-shell nanoparticle size, while retaining near-infrared plasmon resonance, and keeping the nanoparticle surface charge constant, results in a four to five fold tumor accumulation of nanoparticles following equal dose of injected gold for both sizes. The survival time of mice bearing large (>1000mm(3)) and highly aggressive triple negative breast tumors is doubled for the nanomatryushka treatment group under identical photo-thermal therapy conditions. The higher absorption cross-section of a nanomatryoshka results in a higher efficiency of photonic to thermal energy conversion and coupled with 4-5× accumulation within large tumors results in superior therapy efficacy.

Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis.

IntroductionReal-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes.MethodsCTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes.ResultsIn total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients.ConclusionThis study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies. Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition. Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo. Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

Abstract PD01-01: Overcoming endocrine therapy resistance related to PTEN loss by strategic combinations with mTOR, AKT, or MEK inhibitors

Background: Hyperactive PI3K signaling is associated with a more aggressive subtype of estrogen receptor (ER) positive breast cancer (BC) and with endocrine resistance. Loss or downregulation of PI3K9s inhibitor PTEN is more common in basal and luminal B vs. luminal A BC. However, the role of PTEN in modulating response to various endocrine therapies is unclear. Here we investigated the effects of PTEN knockdown (KD) on endocrine sensitivity and the potential of multiple kinase inhibitors to restore and improve responses. Methods: Nude mice bearing ER+ BC xenograft tumors of MCF7 cells stably expressing a doxycycline (Dox)-inducible PTEN-shRNA were randomized to four endocrine treatment groups [continued estrogen (E2) supplementation, or E2-deprivation (ED) alone or in combination with tamoxifen (Tam) or fulvestrant (Ful)]; all -/+ Dox. The effects of single or combined kinase inhibitors on these endocrine treatments -/+ Dox were studied in vitro using inhibitors (i) to mTOR (AZD2014, 0.2 μM), AKT (AZD5363, 1 μM), or MEK (Selumetinib/ARRY-142886, 1 μM). Cell growth, apoptosis, and ER and progesterone receptor (PR) signaling were analyzed using cell cytometry, qRT/PCR, and Western blotting. Synergism tests were used to examine the growth effects of the most promising combinatorial therapy with multiple kinase inhibitors in different endocrine settings. Results: In wild-type (WT) PTEN xenograft tumors, endocrine therapies were very effective, inducing frequent tumor regression. In PTEN KD tumors endocrine therapies were less effective — PTEN KD delayed tumor regression in all endocrine regimens and accelerated tumor progression in the Tam treated group. Furthermore, at day 250, only 1/8 and 0/7 tumors had developed resistance in the ED and the Ful (−Dox) groups, respectively, while with PTEN KD (+Dox), 7/15 and 5/15 tumors developed resistance to ED and to Ful. In vitro PTEN KD also induced resistance to all endocrine therapies. mRNA and/or protein levels of ER and PR were suppressed by PTEN KD and restored by mTORi and AKTi. In cells with WT PTEN, mTORi was highly effective with or without endocrine therapy. However, AKTi and MEKi were more effective in combination with endocrine therapy. All three inhibitors were less effective upon PTEN KD. The mTORi plus AKTi combination resulted in a potent synergistic inhibition in PTEN KD cells in the presence of E2 or with ED. In contrast, in the presence of Tam, AKTi plus MEKi, independent of PTEN status, was the most effective combination at the doses chosen. Finally, these inhibitors and combinations were more effective in the presence of Ful than ED or Tam in WT PTEN cells. AKTi combined with Ful was still highly effective even in PTEN KD cells, but mTORi and MEKi were less effective. Conclusions: Our results suggest that PTEN loss renders endocrine therapy less effective in in vitro and in vivo experimental models. Single AKT/MEK kinase inhibitors are more potent in the presence of endocrine therapy. In PTEN KD cells, the activity of all three kinase inhibitors is largely diminished, except for AKTi in the presence of fulvestrant. Kinase inhibitor combinations are generally more effective, but the optimal combinations vary by PTEN status and type of endocrine therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-01.

Abstract P3-04-07: The new oral SERD AZD9496 is efficacious in antagonizing ER and circumventing resistance to endocrine therapy

Background: The selective estrogen receptor (ER) degrader (SERD) fulvestrant (Ful) is a potent ER antagonist that upon binding to ER induces its degradation. Ful has shown clinical efficacy in metastatic disease upon progression on previous endocrine therapies and superior activitycompared to an aromatase inhibitor as first line therapy when given at a high dose, 500mg. However, major clinical limitations of Ful are its low bioavailability and its route of administration. Here, we assess the efficacy and the mechanism of action of the new oral SERD AZD9496 compared to Ful in our panel of endocrine-sensitive and -resistant (EndoR) in vitro and in vivo models. Methods: The effects of AZD9496 and Ful were studied in vitro in various ER+ MCF7, ZR75-1, T47D, 600MPE, and MDAMB415 parental lines and in MCF7 and T47D derivatives made resistant (R) to estrogen deprivation (ED), tamoxifen (Tam), or Ful. Cell growth, Western blot, Q-RT-PCR, and ERE-reporter assays were conducted to assess treatment efficacy as well as ER levels and activity. Xenografts of parental MCF7 cells were established in ovariectomized nude mice with exogenous estrogen (E2). Mice were then randomized to continued E2 or ED, with and without AZD9496 or Ful. Mice bearing transplantable MCF7 EDR and TamR xenografts were randomized to continue original treatment or to switch to Ful or AZD9496, and tumor size was followed. Expression of classic and nonclassic/indirect ER-regulated genes was evaluated in RNA extracts of short-term-treated xenografts using the BioMark FLUIDIGM platform. Results: AZD9496 inhibited cell growth (50-100%) of all ER+ parental cells and greatly, though not fully, degraded ER protein levels. AZD9496 also potently reduced ER-dependent exogenous and endogenous gene/protein expression in presence and absence of E2. In parental MCF7 xenograft-bearing mice, 10 days of AZD9496 resulted in a greater inhibition of tumor growth and in a greater reduction of levels of ER-dependent targets in comparison to Ful in the presence of E2. The effects of the 2 SERDs were similar in the absence of E2. In EndoR models that retain ER, AZD9496 inhibited cell growth in vitro by degrading ER, similar to Ful. Both SERDs also delayed tumor growth of EDR and TamR xenografts and effectively reduced levels of ER and ER-induced proteins, though no tumor regression was observed in the TamR model. Notably, AZD9496 failed to inhibit growth of FulR cells and xenografts. Expression analysis showed that the 2 SERDs potently inhibited classic ER activity, while simultaneously increasing expression of some genes known to be regulated by the nonclassic/indirect ER activity, including genes involved in escape pathways of endocrine resistance. Conclusions: The oral SERD AZD9496 is a potent antiestrogen that antagonizes and degrades ER. AZD9496, like Ful, inhibits ER-dependent transcription and tumor growth in both naive and resistant EDR and TamR models, but shows cross-resistance in FulR models. Both AZD9496 and Ful failed to completely reduce ER protein expression and to induce TamR tumor regression, suggesting that additional strategies to reduce ER levels and to enhance the inhibition of ER signaling and/or of co-operating survival mechanisms may be needed to improve treatment outcome. Citation Format: Nardone A, Weir H, De Angelis C, Cataldo ML, Fu X, Shea MJ, Mitchell T, Trivedi M, Chamness GC, Osborne CK, Schiff R. The new oral SERD AZD9496 is efficacious in antagonizing ER and circumventing resistance to endocrine therapy [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 P3-04-07.

Abstract P4-01-06: Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis

Introduction: Real-time monitoring of biological changes in tumors that metastasize may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in patients with localized disease pose major hurdles. The goal of this study was to determine if breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes. Methods: CTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counter staining of RBC-lysed blood and bone marrow fractions, respectively. The lung metastasis (LM) rate was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed using the gene expression arrays of primary tumors from different PDX lines and were subsequently overlapped to identify common genes. Results: A total of 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present either as single cells or as clusters, were detected in 83% (15/18) and 62.5% (10/16) of the lines, respectively. There was a positive association between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 out of 18 (50%) lines, of which all 9 had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the 2 genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified 4 genes (HLA-DP1A, GJA1, PEG3, and XIST). This 4-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients. Conclusion: This study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases. Citation Format: Mario Giuliano, Sabrina Herrera, Pavel Christiny, Chad Shaw, Chad J Creighton, Tamika Mitchell, Raksha Bhat, Xiaomei Zhang, Sufeng Mao, Lacey Dobrolecki, Ahmed Al-rawi, Fengju Chen, Bianca M Veneziani, Xiang H Zhang, Susan G Hilsenbeck, Alejandro Contreras, Carolina Gutierrez, Rinath Jeselsohn, Mothaffar F Rimawi, C Kent Osborne, Michael T Lewis, Rachel Schiff, Meghana V Trivedi. Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study 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 P4-01-06.

Abstract P3-05-03: Reversal of endocrine therapy resistance with inhibitors of AKT, mTOR, or MEK as single agents or in combination

Background: Crosstalk between estrogen receptor (ER) and growth factor receptor (GFR) downstream signaling pathways [PI3K/AKT/mTOR and MEK\p42\44MAPK (MAPK)] has been associated with endocrine resistance. Single downstream inhibitors like everolimus partially reverse endocrine resistance. However, more than one downstream escape pathway may contribute to endocrine resistance. Furthermore, disruption by a single downstream inhibitor of the negative feedback loops that balance the amplified signals from GFRs may result in compensatory pathway activation. Therefore we investigated whether dual downstream signaling inhibitions are required to more effectively overcome tamoxifen-resistant growth, using in vitro and in vivo models. Materials and Methods: The effects of different kinase inhibitors (i) AZD2014 (mTORi), AZD5363 (AKTi), and AZD6244 (MEKi) on endocrine therapy [tamoxifen (Tam) and fulvestrant (Fulv)] were tested in ER+ MCF7 and T47D Tam-resistant derivatives (TamR). In vitro growth and apoptosis were assessed using methylene blue and c-PARP, respectively. Western blot analysis was used to analyze the effect of each inhibitor or combination on their respective pathway substrates. Nude mice with transplantable MCF7 TamR xenografts at a size of 200 mm3 were randomized to continued Tam, continued Tam + (mTORi, AKTi, MEKi, mTORi+MEKi, AKTi+MEKi) or Fulv ± the inhibitor combinations. Results: We found that in two ER+ models MCF7 TamR and T47D TamR in vitro, both mTORi and AKTi were effective in restoring growth inhibition, and effective in inhibiting their respective pathways. Interestingly, inhibition of mTOR and AKT resulted in upregulation of pMAPK. However, while MEKi did inhibit its pathway; it did not restore growth inhibition by the antiestrogens. On the other hand, dual inhibition, adding the MEKi to either mTORi or AKTi, resulted in a more potent reduction of cell growth as well as of downstream signaling. In the TamR derivative of MCF7, ER is still maintained and plays a role in resistance, unlike the T47D model, where there is little to no ER expression after TamR develops. Thus, as might be expected, combining downstream inhibitors with potent ER blockade by Fulv enhances the effect of single and dual downstream signaling inhibitors mainly in the MCF7 TamR model. Finally, Fulv in addition to dual downstream inhibitions (mTORi+MEKi or AKTi+MEKi) delayed MCF7 TamR xenograft growth significantly more than Fulv with single downstream inhibitors. Conclusion: This study provides evidence that dual inhibition of GFR downstream pathways is needed to overcome activation of escape mechanisms that are up-regulated with acquired endocrine resistance and after resistance to single pathway inhibitors. Although the downstream inhibitors alone significantly inhibit TamR growth, combination with Fulv robustly slowed growth of TamR tumors in vivo. Based on these results further studies combining MEK inhibition with inhibitors of the PI3K pathway and ER downregulators are warranted. Citation Format: Agostina Nardone, Gladys Morrison, Xiaoyong Fu, Martin Shea, Tamika Mitchell, Teresa Klinowska, C Kent Osborne, Rachel Schiff. Reversal of endocrine therapy resistance with inhibitors of AKT, mTOR, or MEK as single agents or in combination [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 P3-05-03.

Abstract P2-09-08: Therapeutic potential of the dual HER1/2 inhibitor AZD8931 in circumventing endocrine resistance

Background: Crosstalk between ER and HER-family signaling pathways has been suggested to play a role in the development of endocrine resistance. Preclinical studies have shown that AZD8931, a dual tyrosine kinase inhibitor (TKI) of HER1 (EGFR) and HER2, elicits an equipotent inhibition of HER1, HER2, and HER3 signaling, and is consequently more effective in blocking ligand-dependent HER signaling than the dual HER 1/2 TKI lapatinib. Using in vitro and in vivo models we investigated AZD89319s therapeutic potential in enhancing endocrine therapy and in overcoming the growth of tumor cells resistant to tamoxifen (Tam). Materials and Methods: The effect of different TKIs (AZD8931 and lapatinib) on endocrine therapy [Tam and Fulvestrant (Ful)] was tested in ER+ MCF7 and T47D parental cells and their Tam-resistant derivatives (TamRes). In vitro growth, proliferation, and apoptosis were assessed using an in situ cell cytometer, EDU incorporation, and Annexin V-FITC/c-PARP, respectively. HER ligands in the parental and TamRes cells were profiled using RNASeq analysis of these lines. Western blot analysis was used to analyze the effect of AZD8931, lapatinib, and the HER1 TKI gefitinib on HER 1/2 pathway activation upon ligand stimulation. Nude mice with transplantable MCF7/TamRes xenografts at 200 mm 3 were randomized to continued Tam, Tam+AZD8931, Fulvestrant (Ful), or Ful+AZD8931 treatments. Results: We found that neither lapatinib nor AZD8931 significantly enhanced endocrine sensitivity in parental MCF7 breast cancer cells, although AZD8931 did enhance endocrine sensitivity in parental T47D cells. Furthermore, AZD8931 combined with either Tam or Ful inhibited cell growth more profoundly than lapatinib in the T47D TamRes cell model, and was significantly, though modestly, more potent than lapatinib in the MCF7 TamRes model when combined with Tam. In both TamRes models, AZD8931 significantly inhibited cell proliferation and induced apoptosis, with the highest effects seen in combination with Ful. Interestingly, multiple HER ligands are upregulated in both MCF7 and T47D TamRes models, which could explain the superiority of AZD8931 over lapatinib in these models. Indeed, in EGF and heregulin (HRG) stimulated conditions, AZD8931 more potently inhibited HER signaling (i.e., phosphorylated (p) levels of HER1/2, MAPK, and AKT) than lapatinib or gefitinib. Finally, AZD8931 significantly delayed the growth of MCF7 TamRes xenografts in the presence of continued Tam or Ful. These tumors were also inhibited by Ful alone, but the strongest inhibition was achieved by Ful and AZD8931 in combination. However, despite the marked tumor growth delay, no tumor regression was found in any of these treatments. Conclusion: This study provides evidence that AZD8931 has greater inhibitory efficacy than lapatinib in endocrine resistant models that are dependent on ligand activation of the HER pathway. Although the AZD8931 combination with Ful robustly slowed growth of TamRes tumors in vivo , the absence of tumor regression suggests that additional escape pathways are also involved and should also be targeted to fully circumvent tamoxifen resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-08.

P4-01-03: Establishment and Characterization of an Endocrine Resistance Model In Vitro and In Vivo by Inducible PTEN Knockdown.

Background Growth factor receptor and estrogen receptor (ER) are two major driving pathways for initiating and sustaining breast cancer (BC) development and progression. We have previously shown that an inverse correlation exists between the PI3K pathway and ER expression/activity in luminal type BCs. High PI3K activation signaling correlates to the luminal B subtype of BC with low ER expression/activity. However, the involvement of the PI3K pathway tumor suppressor PTEN in resistance to endocrine therapy is less clear. Here we attempt to develop an experimental system to better understand the role of PTEN in this resistance. Materials and Methods : Two luminal BC cell lines, MCF7L and ZR75-B, were stably infected with a lentivirus pINDUCER (Meerbrey et al., PNAS, 2011), containing Tet-on responsive shPTEN, turboRFP (tRFP) as an inducible tag, and enhanced GFP (eGFP) as a constitutive expressed tag for positive cells selection. Immunoblotting of PTEN, phosphorylated (p) Akt, pMAPK, pc-Jun, ER, and ER9s downstream gene products (PR, IGF-1R) was performed on cells after 6 days of doxycycline (Dox) incubation. After pre-starvation for 5 days in estrogen deprivation (ED) conditions, the cells -/+ Dox were subjected to induction with estrogen (E2) or to endocrine treatment [continued ED, tamoxifen (Tam, 10–7M), or fulvestrant (Ful, 10–7M)] in 96-well plate format for 9 days. In situ cell cytometry (Celigo, Cyntellect Inc., San Diego, CA) was applied to count the cell number by scanning the same 96-well plate every other day. Ovariectomized nude mice bearing MCF7L-shPTEN xenografts, established in the presence of E2 supplementation, were randomized to minus and plus Dox groups, with each treatment group continuing E2 or endocrine therapies (ED, Tam, or Ful). The in vivo GFP/RFP imaging was performed with a home-built cooled and image intensified CCD camera system. Results : Immunoblot analysis showed a striking loss of PTEN, and significant upregulation of pAkt, as well as pMAPK and pc-Jun in cells with +Dox. In contrast, levels of ER, PR and IGF1R were reduced in cells with +Dox compared to -Dox. In all -Dox groups, cell growth was significantly reduced in endocrine treated groups compared to the E2 group, whereas +Dox rescued the endocrine treatment growth suppression, especially in ED and Tam groups. In vivo induction of PTEN shRNA expression was confirmed by RFP imaging after feeding the mice with +Dox water. MCF7L-shPTEN xenografts randomized to +Dox under ED conditions continued to grow over 4 weeks, in contrast to the marked regression of control tumors (−Dox) under this endocrine regimen (p Discussion : These data further support the existence of crosstalk between PI3K and ER pathways in luminal type BC. Decreasing PTEN levels by shRNA renders the luminal type BC cells de novo resistant to endocrine therapy both in vitro and in vivo. The pINDUCER PTEN knockdown system combined with live animal imaging offers successful real-time, noninvasive tracking of endocrine sensitivity by controllably manipulating the level of target gene. Combination therapies to overcome endocrine resistance under PTEN knockdown conditions are currently underway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-03.