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mRNA FISH of cell state markers

Supplementary Table 3 from Molecular Characterization of the Transition to Malignancy in a Genetically Engineered Mouse-Based Model of Ductal Carcinoma In situ1

d16HER2 labelled sidebuds. d16HER2 shown in yellow. Krt14 antibody staining shown in green.

Analysis of HER2BOW tumors

p95HER2 invasive carcinoma. p95HER2 shown in magenta. Krt14 antibody staining shown in green.

HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype (WTHER2), exon-16 null (d16HER2), and N-terminally truncated (p95HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. Although WTHER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast, d16HER2 and p95HER2 induced rapid tumor development. d16HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereas p95HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed for d16HER2 and p95HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer.Implications:Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment.

Supplementary Data from Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism

Supplementary Table 2 from Molecular Characterization of the Transition to Malignancy in a Genetically Engineered Mouse-Based Model of Ductal Carcinoma In situ1

Supplementary File 1.

Supplementary Table 1 from Molecular Characterization of the Transition to Malignancy in a Genetically Engineered Mouse-Based Model of Ductal Carcinoma In situ1

UMAP clustering of sequenced cells and cluster identification

TruRegistry of HER2BOW tissues

d16HER2 mammary intraepithelial neoplasm. d16HER2 shown in magenta. Krt14 antibody staining shown in green.

p95HER2 microinvasive carcinoma. p95HER2 shown in magenta. Krt14 antibody staining shown in green.

Supplementary Table 1: Cluster-specific markers from all cells (8,486 cells) Supplementary Table 2: Cluster-specific markers from all epithelial cells (3,843 cells) Supplementary Table 3: Cluster-specific markers from all immune cells (1,803 cells) Supplementary Table 4: Cluster-specific markers from all fibroblast cells (922 cells) Supplementary Table 5a: State-specific markers from all d16:HBOW epithelial cells (1,421 cells) Supplementary Table 5b: State-specific markers from all p95:HBOW epithelial cells (1,258 cells)

Geneset analysis of HER2BOW trajectory mapping

p95HER2 labelled sidebuds. p95HER2 shown in magenta. Krt14 antibody staining shown in green.

A transplantable model of human ductal carcinoma in situ that progresses to invasive carcinoma was developed from a genetically engineered mouse (GEM). Additional lines were established using early mammary premalignant lesions from transgenic MMTV-PyV-mT mice. These lines were verified to be premalignant and transplanted repeatedly to establish stable and predictable properties. Here, we report the first in-depth molecular analysis of neoplastic progression occurring in one premalignant transplantable GEM-derived line. Oligonucleotide microarrays showed that many genes are differentially expressed between the quiescent and prelactating mammary gland and the premalignant GEM outgrowth. In contrast, a small but consistent group of genes was associated with the transformation from premalignancy to tumor. This suggests that the majority of gene expression changes occur during the premalignant transition from normal to premalignancy, whereas many fewer changes occur during the malignant transition from premalignancy to invasive carcinoma. The premalignant transition is associated with several cell cycle–related genes and the up-regulation of oncogenes is associated with various cancers (Ccnd11, Cdk4, Myb, and Ect2). The changes identified in the malignant transition included genes previously associated with human breast cancer progression. Misregulation of the insulin-like growth factor and transforming growth factor-β signaling pathways and the stromal-epithelial interaction were implicated. Our results suggest that this transplantable GEM-based model recapitulates human ductal carcinoma in situ at both histologic and molecular levels. With consistent tumor latency and molecular profiles, this model provides an experimental platform that can be used to assess functional genomics and molecular pharmacology and to test promising chemoprevention strategies.

HER2BOW transgene design

Supplementary Data from Adiponectin Deficiency Limits Tumor Vascularization in the MMTV-PyV-mT Mouse Model of Mammary Cancer

Supplementary Figures S1-S2.

Purpose: High levels of the fat-secreted cytokine adiponectin (APN) are present in the circulation of healthy people, whereas low levels correlate with an increased incidence of breast cancer in women. The current study experimentally probes the physiologic functions of APN in mammary cancer in a newly generated genetic mouse model.Experimental Design: We established an APN null mouse model of mammary cancer by introducing the polyoma virus middle T (PyV-mT) oncogene expressed from mouse mammary tumor virus (MMTV) regulatory elements into APN null mice. MMTV-PyV-mT–induced tumors resemble ErbB2–amplified human breast cancers. We monitored tumor onset, kinetics, and animal survival, and analyzed vascular coverage, apoptosis, and hypoxia in sections from the primary tumors. Metastatic spreading was evaluated by analyses of the lungs.Results: APN prominently localized to the vasculature in human and mouse mammary tumors. In APN null mice, MMTV-PyV-mT–induced tumors appeared with delayed onset and exhibited reduced growth rates. Affected animals survived control tumor-bearing mice by an average of 21 days. Pathologic analyses revealed reduced vascularization of APN null tumors along with increased hypoxia and apoptosis. At the experimental end point, APN null transgenic mice showed increased frequency of pulmonary metastases.Conclusion: The current work identifies a proangiogenic contribution of APN in mammary cancer that, in turn, affects tumor progression. APN interactions with vascular receptors may be useful targets for developing therapies aimed at controlling tumor vascularization in cancer patients.

Purpose: We apply positron emission tomography (PET) to elucidate changes in nanocarrier extravasation during the transition from premalignant to malignant cancer, providing insight into the use of imaging to characterize early cancerous lesions and the utility of nanoparticles in early disease.Experimental Design: Albumin and liposomes were labeled with 64Cu (half-life 12.7 hours), and longitudinal PET and CT imaging studies were conducted in a mouse model of ductal carcinoma in situ. A pharmacokinetic model was applied to estimate the tumor vascular volume and permeability.Results: From early time points characterized by disseminated hyperproliferation, the enhanced vascular permeability facilitated lesion detection. During disease progression, the vascular volume fraction increased 1.6-fold and the apparent vascular permeability to albumin and liposomes increased ∼2.5-fold to 6.6 × 10−8 and 1.3 × 10−8 cm/s, respectively, with the accumulation of albumin increasing earlier in the disease process. In the malignant tumor, both tracers reached similar mean intratumoral concentrations of ∼6% ID/cc but the distribution of liposomes was more heterogeneous, ranging from 1% to 18% ID/cc compared with 1% to 9% ID/cc for albumin. The tumor-to-muscle ratio was 17.9 ± 8.1 and 7.1 ± 0.5 for liposomes and albumin, respectively, indicating a more specific delivery of liposomes than with albumin.Conclusions: PET imaging of radiolabeled particles, validated by confocal imaging and histology, detected the transition from premalignant to malignant lesions and effectively quantified the associated changes in vascular permeability. Clin Cancer Res; 17(3); 550–9. ©2010 AACR.

Supplementary Figure 3 from Distinct ErbB-2–Coupled Signaling Pathways Promote Mammary Tumors with Unique Pathologic and Transcriptional Profiles

PDF file - 7894K, Supplementary Figure 7. Xenograft models of aggressive prostate cancer.

Supplemental Table 2 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplementary Figure 4 from SLITs Suppress Tumor Growth In vivo by Silencing Sdf1/Cxcr4 within Breast Epithelium

Supplementary Figure Legends 1-8 from Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis

XLS file - 58K, List of differentially expressed genes between Nkx3.1CE2/+; Ptenf/f; BrafCA/+ and Nkx3.1CE2/+; Ptenf/f mouse prostate tumors (p-value

Supplementary Figure 2 from Heterogeneous Tumor Evolution Initiated by Loss of pRb Function in a Preclinical Prostate Cancer Model

Supplemental Fig 5 from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Supplementary Table 1 from Distinct ErbB-2–Coupled Signaling Pathways Promote Mammary Tumors with Unique Pathologic and Transcriptional Profiles

XLS file - 188K, List of differentially expressed genes between RAP + PD treated and Vehicle treated Nkx3.1CE2/+; Ptenf/f; Braf CA/+ mouse prostate tumors (p-value

Differential diagnosis and therapy of heterogeneous breast tumors poses a major clinical challenge. To address the need for a comprehensive, noninvasive strategy to define the molecular and functional profiles of tumors in vivo, we investigated a novel combination of metabolic PET and diffusion-weighted (DW)-MRI in the polyoma virus middle T antigen transgenic mouse model of breast cancer. The implementation of a voxelwise analysis for the clustering of intra- and intertumoral heterogeneity in this model resulted in a multiparametric profile based on [18F]Fluorodeoxyglucose ([18F]FDG)-PET and DW-MRI, which identified three distinct tumor phenotypes in vivo, including solid acinar, and solid nodular malignancies as well as cystic hyperplasia. To evaluate the feasibility of this approach for clinical use, we examined estrogen receptor-positive and progesterone receptor-positive breast tumors from five patient cases using DW-MRI and [18F]FDG-PET in a simultaneous PET/MRI system. The postsurgical in vivo PET/MRI data were correlated to whole-slide histology using the latter traditional diagnostic standard to define phenotype. By this approach, we showed how molecular, structural (microscopic, anatomic), and functional information could be simultaneously obtained noninvasively to identify precancerous and malignant subtypes within heterogeneous tumors. Combined with an automatized analysis, our results suggest that multiparametric molecular and functional imaging may be capable of providing comprehensive tumor profiling for noninvasive cancer diagnostics. Cancer Res; 76(18); 5512–22. ©2016 AACR.

PDF file - 103K, Mislocalization of SCRIB enhances EGF-induced AKT activation.

PDF file - 137K, Detailed relationship between the PAP parameter derived through the image-guided model with the classical parameters of solute transport coefficient, clearance, and 30 minute albumin clearance (Cl30).

PDF file - 3402K, Supplementary Figure 6. Additional PTEN/AKT Pathway models.

Detailed description of mean and max value analysis of ADC and [18F]FDG data of tumors of tg(PyV-mT) mice.

PDF file - 51K, Representative biodistribution of tissues 48 hours after nanoparticle injection (N=8). Accumulation was significantly greater for insonified tumors (p

Mean (blue) and maximal (red) ADC and [18F]FDG uptake values of the same representative 8 tumors: VOIs were drawn on the basis of the T2-weighted TSE MRI data, and transferred to the appropriate PET and ADC data. The mean and max values were determined. The comparison between mean or maximal values between the tumors revealed different trends.

Supplementary Figure R from Distinct ErbB-2–Coupled Signaling Pathways Promote Mammary Tumors with Unique Pathologic and Transcriptional Profiles

PDF file - 187K, H&E staining of hyperplasia and tumors from hSCRIBP305L mice.

Supplementary Figure 4 from Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis

Supplementary Figure Legends 1-5 from Tuberous Sclerosis Complex 1: An Epithelial Tumor Suppressor Essential to Prevent Spontaneous Prostate Cancer in Aged Mice

Supplementary Figure Legends 1-6, Table 1 Legend from Fibroblast Growth Factor Receptor Signaling Dramatically Accelerates Tumorigenesis and Enhances Oncoprotein Translation in the Mouse Mammary Tumor Virus–Wnt-1 Mouse Model of Breast Cancer

Supplementary Figure 6 from SLITs Suppress Tumor Growth In vivo by Silencing Sdf1/Cxcr4 within Breast Epithelium

Supplementary Methods, Figure Legends 1-11 from ANCCA/ATAD2 Overexpression Identifies Breast Cancer Patients with Poor Prognosis, Acting to Drive Proliferation and Survival of Triple-Negative Cells through Control of B-Myb and EZH2

Because each change in the evolution of a cancer is predicated on the effects of previous events, a full understanding of selective changes and their effect on tumor progression can only be understood in the context of appropriate initiating events. Here, we define the effect of pRb function inactivation in prostate epithelium on both the initiation of prostate cancer and the establishment of selective pressures that lead to diminished Pten function and tumor evolution. Using genetically engineered mice, we show that inactivation of the pRb family proteins (Rb/p107/p130) induces epithelial proliferation and apoptosis and is sufficient to produce prostatic intraepithelial neoplasia (PIN) lesions. Over time, adenocarcinomas develop in all mice with no evidence of neuroendocrine tumors. Apoptosis is dependent on Pten function and not p53, unlike other epithelial cell types tested previously. Consequently, Pten hemizygosity reduces apoptosis by 50%, accelerating progression to adenocarcinomas with heterogeneous composition. Heterogeneity is associated with concurrent Pten haploinsufficiency and focal selective progression to complete Pten loss, which yields distinct tumor properties. Given that this analysis models the apparent timing of highly penetrant events in human prostate cancer, observed effects may recapitulate the natural evolution of prostate cancer development.

Supplementary Table 1 from Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis