Transformation of enriched mammary cell populations with polyomavirus middle T antigen influences tumor subtype and metastatic potential.

INTRODUCTION: Breast cancer exhibits significant molecular, histological, and pathological diversity. Factors that impact this heterogeneity are poorly understood; however, transformation of distinct normal cell populations of the breast may generate different tumor phenotypes. Our previous study demonstrated that the polyomavirus middle T antigen (PyMT) oncogene can establish diverse tumor subtypes when broadly expressed within mouse mammary epithelial cells. In the present study, we assessed the molecular, histological, and metastatic outcomes in distinct mammary cell populations transformed with the PyMT gene. METHODS: Isolated mouse mammary epithelial cells were transduced with a lentivirus encoding PyMT during an overnight infection and then sorted into hormone receptor-positive luminal (CD133+), hormone receptor-negative luminal (CD133-), basal, and stem cell populations using the cell surface markers CD24, CD49f, and CD133. Each population was subsequently transplanted into syngeneic cleared mouse mammary fat pads to generate tumors. Tumors were classified by histology, estrogen receptor status, molecular subtype, and metastatic potential to investigate whether transformation of different enriched populations affects tumor phenotype. RESULTS: Although enriched mammary epithelial cell populations showed no difference in either the ability to form tumors or tumor latency, differences in prevalence of solid adenocarcinomas and squamous, papillary, and sebaceous-like tumors were observed. In particular, squamous metaplasia was observed more frequently in tumors derived from basal and stem cells than in luminal cells. Interestingly, both molecularly basal and luminal tumors developed from luminal CD133+, basal, and stem cell populations; however, luminal CD133- cells gave rise exclusively to molecularly basal tumors. Tumors arising from the luminal CD133-, basal, and stem cell populations were highly metastatic; however, luminal CD133+ cells generated tumors that were significantly less metastatic, possibly due to an inability of these tumor cells to escape the primary tumor site. CONCLUSIONS: Expression of PyMT within different mammary cell populations influences tumor histology, molecular subtype, and metastatic potential. The data demonstrate that luminal CD133+ cells give rise to less metastatic tumors, luminal CD133- cells preferentially establish basal tumors, and the cell of origin for squamous metaplasia likely resides in the basal and stem cell populations.

Synthesis of Naamidine A and Selective Access to N(2)-Acyl-2-aminoimidazole Analogues.

A short and scalable synthesis of naamidine A, a marine alkaloid with a selective ability to inhibit epidermal growth factor receptor (EGFR)-dependent cellular proliferation, has been achieved. A key achievement in this synthesis was the development of a regioselective hydroamination of a monoprotected propargylguanidine to deliver N(3)-protected cyclic ene-guanidines. This permits the extension of this methodology to prepare N(2)-acyl analogues in a fashion that obviates the troublesome acylation of the free 2-aminoimidazoles, which typically yields mixtures of N(2)- and N(2),N(2)-diacylated products.

Transcription factor Oct1 is a somatic and cancer stem cell determinant.

Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1(HI) cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24(LO)CD44(HI) cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDH(HI) and dye efflux(HI) cells, and increasing Oct1 increases the proportion of ALDH(HI) cells. Normal ALDH(HI) cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function.

On the shoulders of giants: a historical perspective of unique experimental methods in mammary gland research.

While most organs undergo development in utero, the mouse mammary gland orchestrates five major developmental stages following birth: pre-puberty, puberty, pregnancy, lactation, and involution. Induced by both local and systemic factors, these five developmental stages transpire with dramatic alterations in glandular morphology and cellular function. As an experimental system, the mammary gland provides remarkable accessibility to processes regulating stem cell function, hormone response, and epithelial-stromal-extracellular matrix interactions. This review will provide a historical perspective of the unique in vitro and in vivo techniques used to study the mammary gland and how these methods have provided valuable insight into the biology of this organ.

Chemical genetic screen reveals a role for desmosomal adhesion in mammary branching morphogenesis.

During the process of branching morphogenesis, the mammary gland undergoes distinct phases of remodeling to form an elaborate ductal network that ultimately produces and delivers milk to newborn animals. These developmental events rely on tight regulation of critical cellular pathways, many of which are probably disrupted during initiation and progression of breast cancer. Transgenic mouse and in vitro organoid models previously identified growth factor signaling as a key regulator of mammary branching, but the functional downstream targets of these pathways remain unclear. Here, we used purified primary mammary epithelial cells stimulated with fibroblast growth factor-2 (FGF2) to model mammary branching morphogenesis in vitro. We employed a forward chemical genetic approach to identify modulators of this process and describe a potent compound, 1023, that blocks FGF2-induced branching. In primary mammary epithelial cells, we used lentivirus-mediated knockdown of the aryl hydrocarbon receptor (AHR) to demonstrate that 1023 acts through AHR to block branching. Using 1023 as a tool, we identified desmosomal adhesion as a novel target of AHR signaling and show that desmosomes are critical for AHR agonists to block branching. Our findings support a functional role for desmosomes during mammary morphogenesis and also in blocking FGF-induced invasion.

The small molecule C-6 is selectively cytotoxic against breast cancer cells and its biological action is characterized by mitochondrial defects and endoplasmic reticulum stress.

INTRODUCTION: The establishment of drug resistance following treatment with chemotherapeutics is strongly associated with poor clinical outcome in patients, and drugs that target chemoresistant tumors have the potential to increase patient survival. In an effort to identify biological pathways of chemoresistant breast cancers that can be targeted therapeutically, a small molecule screen utilizing metastatic patient-derived breast cancer cells was conducted; from this previous report, the cytotoxic small molecule, C-6, was identified for its ability to selectively kill aggressive breast cancer cells in a caspase-independent manner. Here, we describe the cellular and molecular pathways induced following C-6 treatment in both normal and breast cancer cell lines. METHODS: Transcriptome analyses and protein expression experiments were used to measure endoplasmic reticulum (ER) stress following C-6 treatment. Studies utilizing transmission electron microscopy and metabolomic profiling were conducted to characterize mitochondrial morphology and function in C-6-treated cells. Oxygen consumption rates and oxidative stress were also measured in breast cancer and normal mammary epithelial cells following treatment with the small molecule. Finally, structural modifications were made to the molecule and potency and cancer selectivity were evaluated. RESULTS: Treatment with C-6 resulted in ER stress in both breast cancer cells and normal mammary epithelial cells. Gross morphological defects were observed in the mitochondria and these aberrations were associated with metabolic imbalances and a diminished capacity for respiration. Following treatment with C-6, oxidative stress was observed in three breast cancer cell lines but not in normal mammary epithelial cells. Finally, synthetic modifications made to the small molecule resulted in the identification of the structural components that contribute to C-6's cancer-selective phenotype. CONCLUSIONS: The data reported here implicate mitochondrial and ER stress as a component of C-6's biological activity and provide insight into non-apoptotic cell death mechanisms; targeting biological pathways that induce mitochondrial dysfunction and ER stress may offer new strategies for the development of therapeutics that are effective against chemoresistant breast cancers.

Bis-aryloxadiazoles as effective activators of the aryl hydrocarbon receptor.

Bis-aryloxadiazoles are common scaffolds in medicinal chemistry due to their wide range of biological activities. Previously, we identified a 1,2,4-bis-aryloxadiazole that blocks mammary branching morphogenesis through activation of the aryl hydrocarbon receptor (AHR). In addition to defects in mammary differentiation, AHR stimulation induces toxicity in many other tissues. We performed a structure activity relationship (SAR) study of 1,2,4-bis-aryloxadiazole to determine which moieties of the molecule are critical for AHR activation. We validated our results with a functional biological assay, using desmosome formation during mammary morphogenesis to indicate AHR activity. These findings will aid the design of oxadiazole derivative therapeutics with reduced off-target toxicity profiles.

Development of a screen to identify selective small molecules active against patient-derived metastatic and chemoresistant breast cancer cells.

INTRODUCTION: High failure rates of new investigational drugs have impaired the development of breast cancer therapies. One challenge is that excellent activity in preclinical models, such as established cancer cell lines, does not always translate into improved clinical outcomes for patients. New preclinical models, which better replicate clinically-relevant attributes of cancer, such as chemoresistance, metastasis and cellular heterogeneity, may identify novel anti-cancer mechanisms and increase the success of drug development. METHODS: Metastatic breast cancer cells were obtained from pleural effusions of consented patients whose disease had progressed. Normal primary human breast cells were collected from a reduction mammoplasty and immortalized with human telomerase. The patient-derived cells were characterized to determine their cellular heterogeneity and proliferation rate by flow cytometry, while dose response curves were performed for chemotherapies to assess resistance. A screen was developed to measure the differential activity of small molecules on the growth and survival of patient-derived normal breast and metastatic, chemoresistant tumor cells to identify selective anti-cancer compounds. Several hits were identified and validated in dose response assays. One compound, C-6, was further characterized for its effect on cell cycle and cell death in cancer cells. RESULTS: Patient-derived cells were found to be more heterogeneous, with reduced proliferation rates and enhanced resistance to chemotherapy compared to established cell lines. A screen was subsequently developed that utilized both tumor and normal patient-derived cells. Several compounds were identified, which selectively targeted tumor cells, but not normal cells. Compound C-6 was found to inhibit proliferation and induce cell death in tumor cells via a caspase-independent mechanism. CONCLUSIONS: Short-term culture of patient-derived cells retained more clinically relevant features of breast cancer compared to established cell lines. The low proliferation rate and chemoresistance make patient-derived cells an excellent tool in preclinical drug development.

Breast cancer PDxO cultures for drug discovery and functional precision oncology.

Patient-derived xenografts (PDXs) have clinical value but are time-, cost-, and labor-intensive and thus ill-suited for large-scale experiments. Here, we present a protocol to convert PDX tumors into PDxOs for long-term cultures amenable to moderate-throughput drug screens, including in-depth PDxO validation. We describe steps for PDxO preparation and mouse cell removal. We then detail PDxO validation and characterization and drug response assay. Our PDxO drug screening platform can predict therapy response in vivo and inform functional precision oncology for patients. For complete details on the use and execution of this protocol, please refer to Guillen et al.1.

Identifying biomarkers of differential chemotherapy response in TNBC patient-derived xenografts with a CTD/WGCNA approach.

Although systemic chemotherapy remains the standard of care for TNBC, even combination chemotherapy is often ineffective. The identification of biomarkers for differential chemotherapy response would allow for the selection of responsive patients, thus maximizing efficacy and minimizing toxicities. Here, we leverage TNBC PDXs to identify biomarkers of response. To demonstrate their ability to function as a preclinical cohort, PDXs were characterized using DNA sequencing, transcriptomics, and proteomics to show consistency with clinical samples. We then developed a network-based approach (CTD/WGCNA) to identify biomarkers of response to carboplatin (MSI1, TMSB15A, ARHGDIB, GGT1, SV2A, SEC14L2, SERPINI1, ADAMTS20, DGKQ) and docetaxel (c, MAGED4, CERS1, ST8SIA2, KIF24, PARPBP). CTD/WGCNA multigene biomarkers are predictive in PDX datasets (RNAseq and Affymetrix) for both taxane- (docetaxel or paclitaxel) and platinum-based (carboplatin or cisplatin) response, thereby demonstrating cross-expression platform and cross-drug class robustness. These biomarkers were also predictive in clinical datasets, thus demonstrating translational potential.

Combining the Tyrosine Kinase Inhibitor Cabozantinib and the mTORC1/2 Inhibitor Sapanisertib Blocks ERK Pathway Activity and Suppresses Tumor Growth in Renal Cell Carcinoma.

Current treatment approaches for renal cell carcinoma (RCC) face challenges in achieving durable tumor responses due to tumor heterogeneity and drug resistance. Combination therapies that leverage tumor molecular profiles could offer an avenue for enhancing treatment efficacy and addressing the limitations of current therapies. To identify effective strategies for treating RCC, we selected ten drugs guided by tumor biology to test in six RCC patient-derived xenograft (PDX) models. The multitargeted tyrosine kinase inhibitor (TKI) cabozantinib and mTORC1/2 inhibitor sapanisertib emerged as the most effective drugs, particularly when combined. The combination demonstrated favorable tolerability and inhibited tumor growth or induced tumor regression in all models, including two from patients who experienced treatment failure with FDA-approved TKI and immunotherapy combinations. In cabozantinib-treated samples, imaging analysis revealed a significant reduction in vascular density, and single-nucleus RNA sequencing (snRNA-seq) analysis indicated a decreased proportion of endothelial cells in the tumors. SnRNA-seq data further identified a tumor subpopulation enriched with cell-cycle activity that exhibited heightened sensitivity to the cabozantinib and sapanisertib combination. Conversely, activation of the epithelial-mesenchymal transition pathway, detected at the protein level, was associated with drug resistance in residual tumors following combination treatment. The combination effectively restrained ERK phosphorylation and reduced expression of ERK downstream transcription factors and their target genes implicated in cell-cycle control and apoptosis. This study highlights the potential of the cabozantinib plus sapanisertib combination as a promising treatment approach for patients with RCC, particularly those whose tumors progressed on immune checkpoint inhibitors and other TKIs. SIGNIFICANCE: The molecular-guided therapeutic strategy of combining cabozantinib and sapanisertib restrains ERK activity to effectively suppress growth of renal cell carcinomas, including those unresponsive to immune checkpoint inhibitors.

A pharmacogenomic method for individualized prediction of drug sensitivity.

Identifying the best drug for each cancer patient requires an efficient individualized strategy. We present MATCH (Merging genomic and pharmacologic Analyses for Therapy CHoice), an approach using public genomic resources and drug testing of fresh tumor samples to link drugs to patients. Valproic acid (VPA) is highlighted as a proof-of-principle. In order to predict specific tumor types with high probability of drug sensitivity, we create drug response signatures using publically available gene expression data and assess sensitivity in a data set of >40 cancer types. Next, we evaluate drug sensitivity in matched tumor and normal tissue and exclude cancer types that are no more sensitive than normal tissue. From these analyses, breast tumors are predicted to be sensitive to VPA. A meta-analysis across breast cancer data sets shows that aggressive subtypes are most likely to be sensitive to VPA, but all subtypes have sensitive tumors. MATCH predictions correlate significantly with growth inhibition in cancer cell lines and three-dimensional cultures of fresh tumor samples. MATCH accurately predicts reduction in tumor growth rate following VPA treatment in patient tumor xenografts. MATCH uses genomic analysis with in vitro testing of patient tumors to select optimal drug regimens before clinical trial initiation.

Synthesis and Preliminary Biological Study of Bisindolylmethanes Accessed by an Acid-Catalyzed Hydroarylation of Vinylindoles.

An acid-catalyzed hydroarylation reaction of vinyl indoles is reported, which tolerates a wide range of heterocycles as the exogenous nucleophile such as indoles, pyrroles, and indolizines. The method rapidly accesses the biologically relevant bisindolylmethane scaffold in good to excellent yields. Evaluation of the biological activity of several synthesized analogues reveals cytotoxic activity against and selectivity for the MCF-7 breast cancer cell line.

Multiparametric quantitative phase imaging for real-time, single cell, drug screening in breast cancer.

Quantitative phase imaging (QPI) measures the growth rate of individual cells by quantifying changes in mass versus time. Here, we use the breast cancer cell lines MCF-7, BT-474, and MDA-MB-231 to validate QPI as a multiparametric approach for determining response to single-agent therapies. Our method allows for rapid determination of drug sensitivity, cytotoxicity, heterogeneity, and time of response for up to 100,000 individual cells or small clusters in a single experiment. We find that QPI EC50 values are concordant with CellTiter-Glo (CTG), a gold standard metabolic endpoint assay. In addition, we apply multiparametric QPI to characterize cytostatic/cytotoxic and rapid/slow responses and track the emergence of resistant subpopulations. Thus, QPI reveals dynamic changes in response heterogeneity in addition to average population responses, a key advantage over endpoint viability or metabolic assays. Overall, multiparametric QPI reveals a rich picture of cell growth by capturing the dynamics of single-cell responses to candidate therapies.

Immunologically "cold" triple negative breast cancers engraft at a higher rate in patient derived xenografts.

TNBC is a heterogeneous subtype of breast cancer, and only a subset of TNBC can be established as PDXs. Here, we show that there is an engraftment bias toward TNBC with low levels of immune cell infiltration. Additionally, TNBC that failed to engraft show gene expression consistent with a cancer-promoting immunological state, leading us to hypothesize that the immunological state of the tumor and possibly the state of the immune system of the host may be essential for engraftment.

A path to translation: How 3D patient tumor avatars enable next generation precision oncology.

3D patient tumor avatars (3D-PTAs) hold promise for next-generation precision medicine. Here, we describe the benefits and challenges of 3D-PTA technologies and necessary future steps to realize their potential for clinical decision making. 3D-PTAs require standardization criteria and prospective trials to establish clinical benefits. Innovative trial designs that combine omics and 3D-PTA readouts may lead to more accurate clinical predictors, and an integrated platform that combines diagnostic and therapeutic development will accelerate new treatments for patients with refractory disease.

PDXNet portal: patient-derived Xenograft model, data, workflow and tool discovery.

We created the PDX Network (PDXNet) portal (https://portal.pdxnetwork.org/) to centralize access to the National Cancer Institute-funded PDXNet consortium resources, to facilitate collaboration among researchers and to make these data easily available for research. The portal includes sections for resources, analysis results, metrics for PDXNet activities, data processing protocols and training materials for processing PDX data. Currently, the portal contains PDXNet model information and data resources from 334 new models across 33 cancer types. Tissue samples of these models were deposited in the NCI's Patient-Derived Model Repository (PDMR) for public access. These models have 2134 associated sequencing files from 873 samples across 308 patients, which are hosted on the Cancer Genomics Cloud powered by Seven Bridges and the NCI Cancer Data Service for long-term storage and access with dbGaP permissions. The portal includes results from freely available, robust, validated and standardized analysis workflows on PDXNet sequencing files and PDMR data (3857 samples from 629 patients across 85 disease types). The PDXNet portal is continuously updated with new data and is of significant utility to the cancer research community as it provides a centralized location for PDXNet resources, which support multi-agent treatment studies, determination of sensitivity and resistance mechanisms, and preclinical trials.

A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.

Toward improved models of human cancer.

Human cancer is a complex and heterogeneous collection of diseases that kills more than 18 million people every year worldwide. Despite advances in detection, diagnosis, and treatments for cancers, new strategies are needed to combat deadly cancers. Models of human cancer continue to evolve for preclinical research and have culminated in patient-derived systems that better represent the diversity and complexity of cancer. Still, no model is perfect. This Perspective attempts to address ways that we can improve the clinical translatability of models used for cancer research, from the point of view of researchers who mainly conduct cancer studies in vivo.

Erratum: "Toward improved models of human cancer" [APL Bioeng. 5, 010901 (2021)].

[This corrects the article DOI: 10.1063/5.0030534.].