Characterizing heterogeneous single-cell dose responses computationally and experimentally using threshold inhibition surfaces and dose-titration assays.

Single cancer cells within a tumor exhibit variable levels of resistance to drugs, ultimately leading to treatment failures. While tumor heterogeneity is recognized as a major obstacle to cancer therapy, standard dose-response measurements for the potency of targeted kinase inhibitors aggregate populations of cells, obscuring intercellular variations in responses. In this work, we develop an analytical and experimental framework to quantify and model dose responses of individual cancer cells to drugs. We first explore the connection between population and single-cell dose responses using a computational model, revealing that multiple heterogeneous populations can yield nearly identical population dose responses. We demonstrate that a single-cell analysis method, which we term a threshold inhibition surface, can differentiate among these populations. To demonstrate the applicability of this method, we develop a dose-titration assay to measure dose responses in single cells. We apply this assay to breast cancer cells responding to phosphatidylinositol-3-kinase inhibition (PI3Ki), using clinically relevant PI3Kis on breast cancer cell lines expressing fluorescent biosensors for kinase activity. We demonstrate that MCF-7 breast cancer cells exhibit heterogeneous dose responses with some cells requiring over ten-fold higher concentrations than the population average to achieve inhibition. Our work reimagines dose-response relationships for cancer drugs in an emerging paradigm of single-cell tumor heterogeneity.

Evaluating immunotherapeutic outcomes in triple-negative breast cancer with a cholesterol radiotracer in mice.

Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement for cholesterol in the activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in preclinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and -nonresponsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti-PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells allows detection of T cell activation and has potential to assess the success of ICI therapy.

Broad Next-Generation Integrated Sequencing of Myelofibrosis Identifies Disease-Specific and Age-Related Genomic Alterations.

PURPOSE: Myeloproliferative neoplasms (MPN) are characterized by the overproduction of differentiated myeloid cells. Mutations in JAK2, CALR, and MPL are considered drivers of Bcr-Abl-ve MPN, including essential thrombocythemia (ET), polycythemia vera (PV), prefibrotic primary myelofibrosis (prePMF), and overt myelofibrosis (MF). However, how these driver mutations lead to phenotypically distinct and/or overlapping diseases is unclear. EXPERIMENTAL DESIGN: To compare the genetic landscape of MF to ET/PV/PrePMF, we sequenced 1,711 genes for mutations along with whole transcriptome RNA sequencing of 137 patients with MPN. RESULTS: In addition to driver mutations, 234 and 74 genes were found to be mutated in overt MF (N = 106) and ET/PV/PrePMF (N = 31), respectively. Overt MF had more mutations compared with ET/PV/prePMF (5 vs. 4 per subject, P = 0.006). Genes frequently mutated in MF included high-risk genes (ASXL1, SRSF2, EZH2, IDH1/2, and U2AF1) and Ras pathway genes. Mutations in NRAS, KRAS, SRSF2, EZH2, IDH2, and NF1 were exclusive to MF. Advancing age, higher DIPSS, and poor overall survival (OS) correlated with increased variants in MF. Ras mutations were associated with higher leukocytes and platelets and poor OS. The comparison of gene expression showed upregulation of proliferation and inflammatory pathways in MF. Notably, ADGRL4, DNASE1L3, PLEKHGB4, HSPG2, MAMDC2, and DPYSL3 were differentially expressed in hematopoietic stem and differentiated cells. CONCLUSIONS: Our results illustrate that evolution of MF from ET/PV/PrePMF likely advances with age, accumulation of mutations, and activation of proliferative pathways. The genes and pathways identified by integrated genomics approach provide insight into disease transformation and progression and potential targets for therapeutic intervention.

Growth signaling autonomy in circulating tumor cells aids metastatic seeding.

Self-sufficiency (autonomy) in growth signaling, the earliest recognized hallmark of cancer, is fueled by the tumor cell's ability to "secrete-and-sense" growth factors (GFs); this translates into cell survival and proliferation that is self-sustained by autocrine/paracrine secretion. A Golgi-localized circuitry comprised of two GTPase switches has recently been implicated in the orchestration of growth signaling autonomy. Using breast cancer cells that are either endowed or impaired (by gene editing) in their ability to assemble the circuitry for growth signaling autonomy, here we define the transcriptome, proteome, and phenome of such an autonomous state, and unravel its role during cancer progression. We show that autonomy is associated with enhanced molecular programs for stemness, proliferation, and epithelial-mesenchymal plasticity. Autonomy is both necessary and sufficient for anchorage-independent GF-restricted proliferation and resistance to anticancer drugs and is required for metastatic progression. Transcriptomic and proteomic studies show that autonomy is associated, with a surprising degree of specificity, with self-sustained epidermal growth factor receptor (EGFR)/ErbB signaling. Derivation of a gene expression signature for autonomy revealed that growth signaling autonomy is uniquely induced in circulating tumor cells (CTCs), the harshest phase in the life of tumor cells when it is deprived of biologically available epidermal growth factor (EGF). We also show that autonomy in CTCs tracks therapeutic response and prognosticates outcome. These data support a role for growth signaling autonomy in multiple processes essential for the blood-borne dissemination of human breast cancer.

Interlinked switch circuits of biological intelligence.

Eukaryotic cells learn and adapt via unknown network architectures. Recent work demonstrated a circuit of two GTPases used by cells to overcome growth factor scarcity, encouraging our view that artificial and biological intelligence share strikingly similar design principles and that cells function as deep reinforcement learning (RL) agents in uncertain environments.

Quantitative MRI reveals heterogeneous impacts of treatment on diseased bone marrow in a mouse model of myelofibrosis.

PURPOSE: Analyzing bone marrow in the hematologic cancer myelofibrosis requires endpoint histology in mouse models and bone marrow biopsies in patients. These methods hinder the ability to monitor therapy over time. Preclinical studies typically begin treatment before mice develop myelofibrosis, unlike patients who begin therapy only after onset of disease. Using clinically relevant, quantitative MRI metrics allowed us to evaluate treatment in mice with established myelofibrosis. METHODS: We used chemical shift-encoded fat imaging, DWI, and magnetization transfer sequences to quantify bone marrow fat, cellularity, and macromolecular components in a mouse model of myelofibrosis. We monitored spleen volume, the established imaging marker for treatment, with anatomic MRI. After confirming bone marrow disease by MRI, we randomized mice to treatment with an approved drug (ruxolitinib or fedratinib) or an investigational agent, navitoclax, for 33 days. We measured the effects of therapy over time with bone marrow and spleen MRI. RESULTS: All treatments produced heterogeneous responses with improvements in bone marrow evident in subsets of individual mice in all treatment groups. Reductions in spleen volume commonly occurred without corresponding improvement in bone marrow. MRI revealed patterns associated with effective and ineffective responses to treatment in bone marrow and identified regional variations in efficacy within a bone. CONCLUSIONS: Quantitative MRI revealed modest, heterogeneous improvements in bone marrow disease when treating mice with established myelofibrosis. These results emphasize the value of bone marrow MRI to assess treatment in preclinical models and the potential to advance clinical trials for patients.

Current Status of Cancer Genomics and Imaging Phenotypes: What Radiologists Need to Know.

Ongoing discoveries in cancer genomics and epigenomics have revolutionized clinical oncology and precision health care. This knowledge provides unprecedented insights into tumor biology and heterogeneity within a single tumor, among primary and metastatic lesions, and among patients with the same histologic type of cancer. Large-scale genomic sequencing studies also sparked the development of new tumor classifications, biomarkers, and targeted therapies. Because of the central role of imaging in cancer diagnosis and therapy, radiologists need to be familiar with the basic concepts of genomics, which are now becoming the new norm in oncologic clinical practice. By incorporating these concepts into clinical practice, radiologists can make their imaging interpretations more meaningful and specific, facilitate multidisciplinary clinical dialogue and interventions, and provide better patient-centric care. This review article highlights basic concepts of genomics and epigenomics, reviews the most common genetic alterations in cancer, and discusses the implications of these concepts on imaging by organ system in a case-based manner. This information will help stimulate new innovations in imaging research, accelerate the development and validation of new imaging biomarkers, and motivate efforts to bring new molecular and functional imaging methods to clinical radiology. Keywords: Oncology, Cancer Genomics, Epignomics, Radiogenomics, Imaging Markers Supplemental material is available for this article. © RSNA, 2023.

Predicting efficacy of immunotherapy in mice with triple negative breast cancer using a cholesterol PET radiotracer.

Predicting the response to cancer immunotherapy remains an unmet challenge in triple-negative breast cancer (TNBC) and other malignancies. T cells, the major target of current checkpoint inhibitor immunotherapies, accumulate cholesterol during activation to support proliferation and signaling. The requirement of cholesterol for anti-tumor functions of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged a novel positron emission tomography (PET) radiotracer, FNP-59. FNP-59 is an analog of cholesterol that our group has validated as an imaging biomarker for cholesterol uptake in pre-clinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing immune checkpoint inhibitor (ICI)-responsive EO771 tumors to non-responsive AT-3 tumors, we found significantly higher uptake of a fluorescent cholesterol analog in T cells of the ICI-responsive tumors both in vitro and in vivo. Using the FNP-59 radiotracer, we discovered that accumulation of cholesterol by T cells increased further in ICI-responding tumors that received ant-PD-1 checkpoint immunotherapy. We verified these data by mining single cell sequencing data from patients with TNBC. Patients with tumors containing cycling T cells showed gene expression signatures of cholesterol uptake and trafficking. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells predict T cell activation and success of ICI therapy.

Inhibiting BRAF/EGFR/MEK suppresses cancer stemness and drug resistance of primary colorectal cancer cells.

Drug resistance is a major barrier against successful treatments of cancer patients. Gain of stemness under drug pressure is a major mechanism that renders treatments ineffective. Identifying approaches to target cancer stem cells (CSCs) is expected to improve treatment outcomes for patients. To elucidate the role of cancer stemness in resistance of colorectal cancer cells to targeted therapies, we developed spheroid cultures of patient-derived BRAFmut and KRASmut tumor cells and studied resistance mechanisms to inhibition of MAPK pathway through phenotypic and gene and protein expression analysis. We found that treatments enriched the expression of CSC markers CD166, ALDH1A3, CD133, and LGR5 and activated PI3K/Akt pathway in cancer cells. We examined various combination treatments to block these activities and found that a triple combination against BRAF, EGFR, and MEK significantly reduced stemness and activities of oncogenic signaling pathways. This study demonstrates the feasibility of blocking stemness-mediated drug resistance and tumorigenic activities in colorectal cancer.

Microfluidic single-cell migration chip reveals insights into the impact of extracellular matrices on cell movement.

Cell migration is a complex process that plays a crucial role in normal physiology and pathologies such as cancer, autoimmune diseases, and mental disorders. Conventional cell migration assays face limitations in tracking a large number of individual migrating cells. To address this challenge, we have developed a high-throughput microfluidic cell migration chip, which seamlessly integrates robotic liquid handling and computer vision to swiftly monitor the movement of 3200 individual cells, providing unparalleled single-cell resolution for discerning distinct behaviors of the fast-moving cell population. This study focuses on the ECM's role in regulating cellular migration, utilizing this cutting-edge microfluidic technology to investigate the impact of ten different ECMs on triple-negative breast cancer cell lines. We found that collagen IV, collagen III, and collagen I coatings were the top enhancers of cell movement. Combining these ECMs increased cell motility, but the effect was sub-additive. Furthermore, we examined 87 compounds and found that while some compounds inhibited migration on all substrates, significantly distinct effects on differently coated substrates were observed, underscoring the importance of considering ECM coating. We also utilized cells expressing a fluorescent actin reporter and observed distinct actin structures in ECM-interacting cells. ScRNA-Seq analysis revealed that ECM coatings induced EMT and enhanced cell migration. Finally, we identified genes that were particularly up-regulated by collagen IV and the selective inhibitors successfully blocked cell migration on collagen IV. Overall, the study provides insights into the impact of various ECMs on cell migration and dynamics of cell movement with implications for developing therapeutic strategies to combat diseases related to cell motility.

A hybrid breast cancer/mesenchymal stem cell population enhances chemoresistance and metastasis.

Patients with triple-negative breast cancer remain at risk for metastatic disease despite treatment. The acquisition of chemoresistance is a major cause of tumor relapse and death, but the mechanisms are far from understood. We have demonstrated that breast cancer cells (BCCs) can engulf mesenchymal stem/stromal cells (MSCs), leading to enhanced dissemination. Here, we show that clinical samples of primary invasive carcinoma and chemoresistant breast cancer metastasis contain a unique hybrid cancer cell population coexpressing pancytokeratin and the MSC marker fibroblast activation protein-α. We show that hybrid cells form in primary tumors and that they promote breast cancer metastasis and chemoresistance. Using single-cell microfluidics and in vivo models, we found that there are polyploid senescent cells within the hybrid cell population that contribute to metastatic dissemination. Our data reveal that Wnt Family Member 5A (WNT5A) plays a crucial role in supporting the chemoresistance properties of hybrid cells. Furthermore, we identified that WNT5A mediates hybrid cell formation through a phagocytosis-like mechanism that requires BCC-derived IL-6 and MSC-derived C-C Motif Chemokine Ligand 2. These findings reveal hybrid cell formation as a mechanism of chemoresistance and suggest that interrupting this mechanism may be a strategy in overcoming breast cancer drug resistance.

Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system.

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust ßarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced ßarr-coupling compared to CXCR7. These two receptors induce distinct ßarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for ßarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery.

Recurrent and convolutional neural networks for sequential multispectral optoacoustic tomography (MSOT) imaging.

Multispectral optoacoustic tomography (MSOT) is a beneficial technique for diagnosing and analyzing biological samples since it provides meticulous details in anatomy and physiology. However, acquiring high through-plane resolution volumetric MSOT is time-consuming. Here, we propose a deep learning model based on hybrid recurrent and convolutional neural networks to generate sequential cross-sectional images for an MSOT system. This system provides three modalities (MSOT, ultrasound, and optoacoustic imaging of a specific exogenous contrast agent) in a single scan. This study used ICG-conjugated nanoworms particles (NWs-ICG) as the contrast agent. Instead of acquiring seven images with a step size of 0.1 mm, we can receive two images with a step size of 0.6 mm as input for the proposed deep learning model. The deep learning model can generate five other images with a step size of 0.1 mm between these two input images meaning we can reduce acquisition time by approximately 71%.

Enhanced mitochondrial fission inhibits triple-negative breast cancer cell migration through an ROS-dependent mechanism.

Mitochondria produce reactive oxygen species (ROS), which function in signal transduction. Mitochondrial dynamics, encompassing morphological shifts between fission and fusion, can directly impact ROS levels in cancer cells. In this study, we identified an ROS-dependent mechanism for how enhanced mitochondrial fission inhibits triple negative breast cancer (TNBC) cell migration. We found that enforcing mitochondrial fission in TNBC resulted in an increase in intracellular ROS levels and reduced cell migration and the formation of actin-rich migratory structures. Consistent with mitochondrial fission, increasing ROS levels in cells inhibited cell migration. Conversely, reducing ROS levels with either a global or mitochondrially targeted scavenger overcame the inhibitory effects of mitochondrial fission. Mechanistically, we found that the ROS sensitive SHP-1/2 phosphatases partially regulate inhibitory effects of mitochondrial fission on TNBC migration. Overall, our work reveals the inhibitory effects of ROS in TNBC and supports mitochondrial dynamics as a potential therapeutic target for cancer.

Triple-negative breast cancer cells invade adipocyte/preadipocyte-encapsulating geometrically inverted mammary organoids.

This paper describes the manufacture of geometrically inverted mammary organoids encapsulating primary mammary preadipocytes and adipocytes. Material manipulation in an array of 192 hanging drops induces cells to self-assemble into inside-out organoids where an adipose tissue core is enveloped by a cell-produced basement membrane, indicated by laminin V staining and then a continuous layer of mammary epithelial cells. This inverted tissue structure enables investigation of multiple mammary cancer subtypes, with a significantly higher extent of invasion by triple-negative MDA-MB-231 breast cancer cells compared to MCF7 cells. By seeding cancer cells into co-culture around pre-formed organoids with encapsulated preadipocytes/adipocytes, invasion through the epithelium, then into the adipose core is observable through acquisition of confocal image stacks of whole mount specimens. Furthermore, in regions of the connective tissue core where invasion occurs, there is an accumulation of collagen in the microenvironment. Suggesting that this collagen may be conducive to increased invasiveness, the anti-fibrotic drug pirfenidone shows efficacy in this model by slowing invasion. Comparison of adipose tissue derived from three different donors shows method consistency as well as the potential to evaluate donor cell-based biological variability. Insight box Geometrically inverted mammary organoids encapsulating primary preadipocytes/adipocytes (P/As) are bioengineered using a minimal amount of Matrigel scaffolding. Use of this eversion-free method is key to production of adipose mammary organoids (AMOs) where not only the epithelial polarity but also the entire self-organizing arrangement, including adipose position, is inside-out. While an epithelial-only structure can analyze cancer cell invasion, P/As are required for invasion-associated collagen deposition and efficacy of pirfenidone to counteract collagen deposition and associated invasion. The methods described strike a balance between repeatability and preservation of biological variability: AMOs form consistently across multiple adipose cell donors while revealing cancer cell invasion differences.