Supplementary Figure Legend from Three-Dimensional Imaging and Quantification of Both Solitary Cells and Metastases in Whole Mouse Liver by Magnetic Resonance Imaging

COPYRIGHT © 2023 Santiago-Gómez, Barkan and Chambers. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Editorial PUBLISHED 30 January 2023 DOI 10.3389/fonc.2023.1126924

Supplementary Table 1, Figures 1-4 from Notch1 Inhibition Alters the CD44hi/CD24lo Population and Reduces the Formation of Brain Metastases from Breast Cancer

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS‐like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high‐resolution, isoform‐specific context, we conducted genome‐wide chromatin‐immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial–mesenchymal transition (EMT)‐related genes, including SLUG and TWIST1 . Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3‐induced migration and invasion. Assessing TBX3 by immunohistochemistry in early‐stage (stage 0 and stage I) breast cancers revealed high expression in low‐grade lesions. Within a second independent early‐stage non‐high‐grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo . These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early‐stage pre‐invasive breast cancer to invasive carcinoma through the low‐grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

TBX3 is a member of the highly conserved family of T-box transcription factors involved in embryogenesis, organogenesis and tumor progression. While the functional role of TBX3 in tumorigenesis has been widely studied, less is known about the specific functions of the different isoforms (TBX3iso1 and TBX3iso2) which differ in their DNA-binding domain. We therefore sought to investigate the functional consequence of this highly conserved splice event as it relates to TBX3-induced tumorigenesis. By utilizing a nude mouse xenograft model, we have identified differential tumorigenic potential between TBX3 isoforms, with TBX3iso1 overexpression more commonly associated with invasive carcinoma and high tumor vascularity. Transcriptional analysis of signaling pathways altered by TBX3iso1 and TBX3iso2 overexpression revealed significant differences in angiogenesis-related genes. Importantly, osteopontin (OPN), a cancer-associated secreted phosphoprotein, was significantly up-regulated with TBX3iso1 (but not TBX3iso2) overexpression. This pattern was observed across three non/weakly-tumorigenic breast cancer cell lines (21PT, 21NT, and MCF7). Up-regulation of OPN in TBX3iso1 overexpressing cells was associated with induction of hyaluronan synthase 2 (HAS2) expression and increased retention of hyaluronan in pericellular matrices. These transcriptional changes were accompanied by the ability to induce endothelial cell vascular channel formation by conditioned media in vitro, which could be inhibited through addition of an OPN neutralizing antibody. Within the TCGA breast cancer cohort, we identified an 8.1-fold higher TBX3iso1 to TBX3iso2 transcript ratio in tumors relative to control, and this ratio was positively associated with high-tumor grade and an aggressive molecular subtype. Collectively, the described changes involving TBX3iso1-dependent promotion of angiogenesis may thus serve as an adaptive mechanism within breast cancer cells, potentially explaining differences in tumor formation rates between TBX3 isoforms in vivo. This study is the first of its kind to report significant functional differences between the two TBX3 isoforms, both in vitro and in vivo.

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.

During contrast-enhanced ultrasound (CEUS) imaging of tumor microvascular perfusion, nearby arteriole enhancement can be a dominant feature of the wash-in kinetics that obscures the perfusion characteristics of the capillary bed. This presentation demonstrates that statistical wash-in curves generated using our compound CEUS speckle model1 exhibit two distinct phases corresponding to “fast-flow” and “slow-flow” enhancement that can be detected by fitting a linear combination of two monoexponential functions with different time constants. CEUS cine loops were acquired from a patient-derived xenograft model of renal cell carcinoma, where fresh tumor fragments were engrafted into the chicken embryo chorioallantoic membrane. Subharmonic CEUS images were acquired at 18 MHz using a destruction-replenishment protocol. Enhancement of manually segmented tumor cross-sections was analyzed offline in MATLAB. The CEUS cine loops from this xenograft model frequently exhibited in-plane arteriole enhancement. The two-phase fit discriminated a slow-flow component in 26.6% of time-intensity wash-in curves and 62.9% of the statistical wash-in curves, leading to a decrease in estimated tumor blood volume by 22.1% and 24.7%, respectively, compared to a simple monoexponential fit. These results suggest that conventional CEUS processing may frequently overestimate capillary blood volume. 1M.R. Lowerison et al., Med. Phys. 44, 99-111 (2017).

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2018MP72-14 IDENTIFYING THE SPATIO-FUNCTIONAL ORIGINS OF DRUG RESISTANCE WITH RAPID TUMOR XENOGRAFTS Nicholas Power, Matthew Lowerison, Yaroslav Fedyshyn, Karla Williams, Ann Chambers, James Lacefield, Paul Boutros, and Hon Leong Nicholas PowerNicholas Power More articles by this author , Matthew LowerisonMatthew Lowerison More articles by this author , Yaroslav FedyshynYaroslav Fedyshyn More articles by this author , Karla WilliamsKarla Williams More articles by this author , Ann ChambersAnn Chambers More articles by this author , James LacefieldJames Lacefield More articles by this author , Paul BoutrosPaul Boutros More articles by this author , and Hon LeongHon Leong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2298AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Treatment of patients with advanced cancers increasingly relies on expensive agents targeting specific molecular or cellular aberrations. Pre-existing and acquired drug resistance typically renders these therapies ineffective, leading to lethal disease. The genome and microenvironment of cancers vary spatially, allowing drug resistance to emerge in any tumor region. To quantify this spatio-functional heterogeneity in response to therapy, we developed an approach based on tumor-implantation into the chorioallantoic membrane of chick embryos (PDXovo). METHODS Various cores were obtained from the primary tumor and metastases from patients with metastatic renal cell carcinoma at the time of nephrectomy (N=6). Each of these cores (5-6 for the primary tumor and 1-3 for metastases) were subdivided into 3 mm sized fragments and were implanted into the chorioallantoic membrane of chick embryos at Day 9 of embryonic development. Two days later, PDXs were treated topically with vehicle or Sunitinib (10 uM final in DMSO) every day until Day 17 of embryonic development. At endpoint (Day 19), all PDXs were submitted to high-frequency ultrasound imaging to quantitate differences in tumor blood perfusion and tumor volume between treatment groups. After imaging, PDXs were individually submitted to total exome sequencing. RESULTS We apply this approach to 1548 tumor regions from six renal cell carcinoma patients, achieving a 93.6% engraftment rate. We quantify the spatial heterogeneity in response of these models to sunitinib, an anti-angiogenic therapy, and predict clinical resistance using ultrasound imaging. Combining functional and somatic genomics of the primary tumor and metastases revealed some mutational features associated with sunitinib resistance both at baseline and after treatment. CONCLUSIONS These studies establish a rapid and efficient new method of surveying functional tumor heterogeneity in the context of drug resistance. Within 8-10 days of drug challenge, various regions of the primary tumor and metastases can be determined to be resistant or sensitive to targeted therapy, with no outright identifiable molecular signature correlated to drug resistance. This phenotype-based readout was superior to various prognostic scoring criteria systems and point towards PDX based methods to predict de novo drug resistance in patients with metastatic renal cell carcinoma. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e957 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Nicholas Power More articles by this author Matthew Lowerison More articles by this author Yaroslav Fedyshyn More articles by this author Karla Williams More articles by this author Ann Chambers More articles by this author James Lacefield More articles by this author Paul Boutros More articles by this author Hon Leong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Additional file 2. External file sources used in study.

Additional file 7. Gene-list of targets which bind TDG and are regulated by E2.

OBJECTIVE:Effective targeted therapies for patients with triple-negative breast cancer (TNBC) present an unmet clinical need. There is evidence that TNBCs often have increased expression of the epidermal growth factor receptor (EGFR) and of osteopontin (OPN). OPN-mediated signaling can activate EGFR-dependent signaling pathways. Here, we assessed OPN as a potential predictive biomarker for response to anti-EGFR therapy in TNBC.RESEARCH DESIGN AND METHODS:Using two different TNBC cell lines, MDA-MB-468 and MDA-MB-231, we investigated the impact of stable expression of OPN on efficacy of the EGFR inhibitor erlotinib in vitro.RESULTS:We observed that breast cancer cells engineered to overexpress OPN are more sensitive to growth inhibition by erlotinib than control cells. The level of response was related to the level of OPN expression, possibly due to increased phosphorylation status of EGFR Tyr1068.CONCLUSIONS:These results indicate that OPN expression levels are related to sensitivity of TNBC cells to growth inhibition by erlotinib. OPN thus is a promising predictive biomarker for anti-EGFR therapy in breast cancer.

Studying the complex mechanisms underlying breast cancer metastasis and therapy response necessitates relevant in vivo models, particularly syngeneic models with an intact immune system. Two syngeneic spontaneously metastatic sublines, D2A1-m1 and D2A1-m2, were generated from the poorly metastasising BALB/c-derived D2A1 cell line by serial in vivo passaging. In vivo and in vitro analyses revealed distinct and shared characteristics of the metastatic D2A1-m1 and D2A1-m2 sublines. In particular, D2A1-m1 cells are more aggressive in experimental metastasis assays, while D2A1-m2 cells are more efficient at disseminating from the primary tumour in spontaneous metastasis assays. Surprisingly, classical metastasis-associated in vitro phenotypes, such as enhanced proliferation, migration and invasion, are reduced in the sublines compared to the parental cell line. Further, evasion of immune control cannot fully explain their enhanced metastatic properties. By contrast, both sublines show increased resistance to apoptosis when cultured in non-adherent conditions and, for the D2A1-m2 subline, increased 3D tumour spheroid growth. Moreover, the enhanced spontaneous metastatic phenotype of the D2A1-m2 subline is associated with an increased ability to recruit an activated tumour stroma. The metastatic D2A1-m1 and D2A1-m2 cell lines provide additional syngeneic models for investigating the different steps of the metastatic cascade and thereby represent valuable tools for breast cancer researchers. Finally, this study highlights that morphology and cell behaviour in 2D cell-based assays cannot be used as a reliable predictor of metastatic behaviour in vivo.

Brain metastasis is becoming increasingly prevalent in breast cancer due to improved extra-cranial disease control. With emerging availability of modern image-guided radiation platforms, mouse models of brain metastases and small animal magnetic resonance imaging (MRI), we examined brain metastases’ responses from radiotherapy in the pre-clinical setting. In this study, we employed half brain irradiation to reduce inter-subject variability in metastases dose-response evaluations.

Background The estrogen receptor (ER) is a ligand-dependant transcription factor expressed in many breast cancers and is the target of many endocrine-based cancer therapies. Genome-wide studies have shown that the ER binds to gene-specific enhancer regions in response to β-estradiol (E2) which undergo transcription producing noncoding enhancer RNA (eRNA). While eRNAs are important for transcriptional activation of neighboring genes, the mechanism remains poorly understood. Results Using ChIP-Seq we generate a global profile of thymine DNA glycosylase (TDG), an ER coactivator that plays an essential role in DNA demethylation, in response to E2 in the MCF7 breast cancer cell line. Remarkably, we found that in response to E2 TDG localized to enhancers which also recruit ERα, RNA Pol II and other coregulators and which are marked by histone modifications indicative of active enhancers. Importantly, depletion of TDG inhibits E2-mediated transcription of eRNAs and transcription of ER-target genes. Functionally, we find that TDG both sensitizes MCF7 cells to tamoxifen-mediated cytostasis and increases migration and invasion of MCF7 cells. Conclusions Taken together we find that TDG plays a central role in mediating transcription at a subset of enhancers and governs how MCF7 cells respond to both estrogenic and anti-estrogenic compounds and may be an effective therapeutic target.

Purpose: To evaluate whether concurrent neoadjuvant radiation added to standard chemotherapy could increase the pathologic complete response (pCR) to treatment for locally advanced breast cancer (LABC). Methods and Materials: This prospective phase 2 trial recruited 32 LABC patients from 2009 to 2011. Patients received neoadjuvant every-3-weekly 5-fluorouracil (500 mg/m(2)), epirubicin (100 mg/m(2)), and cyclophosphamide (500 mg/m(2)) for 3 cycles, followed by weekly docetaxel (35 mg/m(2)) for 9 cycles. Regional radiation (45 Gy/25 plus 5.4 Gy/5) was delivered concurrently with docetaxel, then modified radical mastectomy. Patients were matched post hoc by a blinded statistician to a concurrent cohort treated with neoadjuvant chemotherapy, modified radical mastectomy, and adjuvant regional radiation. Results: Thirty of 32 patients completed treatment. Twenty-seven were successfully matched by propensity score to 81 control patients by age, stage, and molecular subtype. The concurrent chemoradiation produced a significant increase in pCR (14% vs 22%, P<. 001) but no statistically significant difference in disease-free and overall survival at 3 years (respectively, 69% vs 81%, PZ. 186, hazard ratio 0.51; and 74% vs 89%, PZ. 162, hazard ratio 0.46). Toxicity included 25% of patients with grade 3 pneumonitis and 25% of patients with dermatitis, and 1 death. Conclusions: Concurrent neoadjuvant radiation added to radiosensitizing chemotherapy significantly improved pCR. A prospective randomized clinical trial is warranted to exploit the improved response seen with concurrent therapy but using another radio-sensitizing taxane, to better minimize treatment-related toxicity and determine its impact on overall survival. (C) 2017 Elsevier Inc. All rights reserved.

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2017MP73-06 DRUG RESISTANCE CONSEQUENCES OF TUMOR HETEROGENEITY IN METASTATIC RENAL CELL CARCINOMA USING ULTRA-FAST PATIENT DERIVED XENOGRAFTS AND MULTIREGIONAL GENOMIC SEQUENCING Matt Lowerison, Slavic Fedyshyn, Stephenie Prokopec, Paul Boutros, Ann Chambers, James Lacefield, Nicholas Power, and Hon Leong Matt LowerisonMatt Lowerison More articles by this author , Slavic FedyshynSlavic Fedyshyn More articles by this author , Stephenie ProkopecStephenie Prokopec More articles by this author , Paul BoutrosPaul Boutros More articles by this author , Ann ChambersAnn Chambers More articles by this author , James LacefieldJames Lacefield More articles by this author , Nicholas PowerNicholas Power More articles by this author , and Hon LeongHon Leong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3351AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Next generation preclinical models of renal cell carcinoma (RCC) now offer the ability to pre-determine de novo drug resistance in fresh patient tumor samples prior to targeted therapy. Implantation of tumor specimens into the chorioallantoic membrane (CAM) of the chicken embryo results in high engraftment efficiencies within two days, permitting large scale ″tumor avatar″ studies. Functional tumor heterogeneity studies, which can be performed in context of drug resistance within two weeks, can guide the selection of drugs and predict drug resistance outcomes for RCC patients. This ultrafast PDX model is mirrored by high-frequency ultrasound imaging that permits quantification of tumor volume and tumor vascularity in a high-throughput manner. METHODS Several core biopsies were extracted from primary tumors and metastases from clear cell RCC, chromophobe RCC, and type 1/2 papillary RCC patients and submitted to xenografting into the CAM of chick embryos. At least 6 regions of the primary tumor were xenografted and 3 metastases were xenografted. At least N>36 per region was submitted to xenografting with half of these treated with sunitinib or vehicle (DMSO). At T=10 days post-implantation, high frequency ultrasound imaging was used to quantitate tumor vascularity, tumor volume, tumor blood flow and and tumor blood volume. After imaging, total exome sequencing was performed to identify any genetic mutations for correlation to drug resistance. RESULTS Using this ″tumor avatar″ model paired with a prospective RCC patient cohort, we observe intratumoral functional heterogeneity in the context of sunitinib treatment, as determined by high-frequency ultrasound imaging, highlighting its potential interventional role in the clinic. Exome sequencing and gene copy number variation analysis did not reveal DNA mutation signatures that were associated with resistance to sunitinib treatment within this intratumoral set of PDX biopsies. CONCLUSIONS These findings suggest that genetic tumor heterogeneity exists, but evidence for a direct relationship to the drug resistant phenotype was not manifest in DNA mutations. Therefore, these results support a phenotype based readout to predict drug resistance within 10 days as opposed to a genotype signature, and that drug resistance to targeted therapy is heterogeneous across the primary tumor. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e965 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Matt Lowerison More articles by this author Slavic Fedyshyn More articles by this author Stephenie Prokopec More articles by this author Paul Boutros More articles by this author Ann Chambers More articles by this author James Lacefield More articles by this author Nicholas Power More articles by this author Hon Leong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Purpose: This paper proposes a method for analyzing the first‐order speckle statistics of nonlinear contrast‐enhanced ultrasound images from tumors. Methods: Contrast signal intensity is modeled as a compound distribution of exponential probability density functions with a gamma weighting function. The gamma probability weighting function serves as an approximation for log‐normally distributed flow velocities in a vascular network. The model was applied to sub‐harmonic bolus‐injection images acquired from a mouse breast cancer xenograft model treated with murine version bevacizumab. Results: The area under curve produced using the compound statistical model could more accurately discriminate anti‐VEGF‐treated tumors from untreated tumors than conventional contrast‐enhanced ultrasound image processing. This result was validated with gold standard histological measures of microvascular density. Fractal vessel geometry was estimated using the gamma weighting function and tested against micro‐CT perfusion casting. Treated tumors had a significantly lower vascular fractal dimension than control tumors. Vascular complexity estimated using the ultrasound compound statistical model performed similarly to micro‐CT fractal dimension for discriminating treated from control tumors. Conclusion: The proposed technique can quantify tumor perfusion and provide an index of vascular complexity, making it a potentially useful addition for clinical detection of vascular normalization in anti‐angiogenic trials.

Contrast-enhanced ultrasound (CEUS) permits quantification and monitoring of tumor vascular changes in response to anti-angiogenic treatment with the goal of informing targeted therapy. Conventional mean-intensity-based CEUS analysis discounts additional information that may be available from the first-order speckle statistics in a CEUS image. We demonstrate that our compound speckle model for analysis of CEUS images reduces the variability in tumor perfusion quantification, particularly for estimates of blood volume, and reduces the sensitive/resistant classification ambiguity caused by heterogeneous tumor samples in a patient-derived xenograft model of renal cell carcinoma.

Cancer cell 'invasiveness' is one of the main driving forces in cancer metastasis, and assays that quantify this key attribute of cancer cells are crucial in cancer metastasis research. The research goal of many laboratories is to elucidate the signaling pathways and effectors that are responsible for cancer cell invasion, but many of these experiments rely on in vitro methods that do not specifically simulate individual steps of the metastatic cascade. Cancer cell extravasation is arguably the most important example of invasion in the metastatic cascade, whereby a single cancer cell undergoes transendothelial migration, forming invasive processes known as invadopodia to mediate translocation of the tumor cell from the vessel lumen into tissue in vivo. We have developed a rapid, reproducible and economical technique to evaluate cancer cell invasiveness by quantifying in vivo rates of cancer cell extravasation in the chorioallantoic membrane (CAM) of chicken embryos. This technique enables the investigator to perform well-powered loss-of-function studies of cancer cell extravasation within 24 h, and it can be used to identify and validate drugs with potential antimetastatic effects that specifically target cancer cell extravasation. A key advantage of this technique over similar assays is that intravascular cancer cells within the capillary bed of the CAM are clearly distinct from extravasated cells, which makes cancer cell extravasation easy to detect. An intermediate level of experience in injections of the chorioallantoic membrane of avian embryos and cell culture techniques is required to carry out the protocol.