Retrograde tracing of breast cancer-associated sensory neurons.

Breast cancer is one of the leading causes of mortality among women. The tumour microenvironment, consisting of host cells and extracellular matrix, has been increasingly studied for its interplay with cancer cells, and the resulting effect on tumour progression. While the breast is one of the most innervated organs in the body, the role of neurons, and specifically sensory neurons, has been understudied, mostly for technical reasons. One of the reasons is the anatomy of sensory neurons: sensory neuron somas are located in the spine, and their axons can extend longer than a meter across the body to provide innervation in the breast. Next, neurons are challenging to culture, and there are no cell lines adequately representing the diversity of sensory neurons. Finally, sensory neurons are responsible for transporting several different types of signals to the brain, and there are many different subtypes of sensory neurons. The subtypes of sensory neurons, which innervate and interact with breast tumours, are unknown. To establish the tools for labelling and subtyping neurons that interact with breast cancer cells, we utilised two retrograde tracer's standards in neuroscience, wheat-germ agglutinin (WGA) and cholera toxin subunit B (CTB). In vitro, we employed primary sensory neurons isolated from mouse dorsal root ganglia, cultured in a custom-built microfluidic device DACIT, that mimics the anatomical compartmentalisation of the sensory neuron's soma and axons. In vivo, we utilised both syngeneic and transgenic mouse models of mammary carcinoma. We show that CTB and WGA trace different but overlapping sensory neuronal subpopulations: while WGA is more efficient in labelling CGRP+ neurons, CTB is superior in labelling the NF200+ neurons. Surprisingly, both tracers are also taken up by a significant population of breast cancer cells, both in vitro and in vivo. In summary, we have established methodologies for retrograde tracing of sensory neurons interacting with breast cancer cells. Our tools will be useful for future studies of breast tumour innervation, and development of therapies targeting breast cancer-associated neuron subpopulations of sensory neurons. Lay description: Breast cancer is an aggressive disease that affects both women and men throughout the world. While it has been reported that the increasing size of nerves in breast cancer correlates to bad prognosis in patients, the role of nerves, especially sensory nerves, in breast cancer progression, has remained largely understudied. Sensory nerves are responsible for delivering signals such as pain, mechanical forces (pressure, tension, stretch, touch) and temperature to the brain. The human body is densely innervated, and nerves extending into peripheral organs can be as long as a few meters. Nerve classification and function can be very complex, as they contain bundles of extensions (axons) originating in different neuronal bodies (soma). Maintaining neurons and growing axons in cell culture conditions in order to mimic innervation is technically challenging, as it involves multiple organs of the human body. Here, we focus on tracing sensory axons from the breast tumours back to the neuronal soma, located in the dorsal root ganglia, inside the spine. To do so, we are using two different 'retrograde' tracers, WGA and CTB, which are proteins with a natural ability to enter axons and travel in a retrograde fashion, arriving at the soma, even if it means to travel distances longer than a meter. Both tracers are fluorescently labelled, making them visible using high-resolution fluorescent microscopy. We show that both WGA and CTB can label sensory neurons in tumours, or in cell culture conditions. The two tracers differ in efficiency of tracing different sensory neurons subpopulations: while WGA is more efficient in tracing small C-fibres (CGRP-positive), CTB is more efficient in tracing A-fibres (NF200+) of sensory neurons. In summary, we have successfully established retrograde tracing techniques for sensory neurons towards studying and targeting breast cancer innervation.

Invadopodia-mediated ECM degradation is enhanced in the G1 phase of the cell cycle.

The process of tumor cell invasion and metastasis includes assembly of invadopodia, protrusions capable of degrading the extracellular matrix (ECM). The effect of cell cycle progression on invadopodia has not been elucidated. In this study, by using invadopodia and cell cycle fluorescent markers, we show in 2D and 3D cultures, as well as in vivo, that breast carcinoma cells assemble invadopodia and invade into the surrounding ECM preferentially during the G1 phase. The expression (MT1-MMP, also known as MMP14, and cortactin) and localization (Tks5; also known as SH3PXD2A) of invadopodia components are elevated in G1 phase, and cells synchronized in G1 phase exhibit significantly higher ECM degradation compared to the cells synchronized in S phase. The cyclin-dependent kinase inhibitor (CKI) p27kip1 (also known as CDKN1B) localizes to the sites of invadopodia assembly. Overexpression and stable knockdown of p27kip1 lead to contrasting effects on invadopodia turnover and ECM degradation. Taken together, these findings suggest that expression of invadopodia components, as well as invadopodia function, are linked to cell cycle progression, and that invadopodia are controlled by cell cycle regulators. Our results caution that this coordination between invasion and cell cycle must be considered when designing effective chemotherapies.

ECM Cross-Linking Regulates Invadopodia Dynamics.

Invadopodia are membrane protrusions dynamically assembled by invasive cancer cells in contact with the extracellular matrix (ECM). Invadopodia are enriched by the structural proteins actin and cortactin as well as metalloproteases such as MT1-MMP, whose function is to degrade the surrounding ECM. During metastasis, invadopodia are necessary for cancer cell intravasation and extravasation. Although signaling pathways involved in the assembly and function of invadopodia are well studied, few studies address invadopodia dynamics and how the cell-ECM interactions contribute to cell invasion. Using iterative analysis based on time-lapse microscopy and mathematical modeling of invasive cancer cells, we found that cells oscillate between invadopodia presence and cell stasis-termed the "invadopodia state"-and invadopodia absence during cell translocation-termed the "migration state." Our data suggest that ß1-integrin-ECM binding and ECM cross-linking control the duration of each of the two states. By changing the concentration of cross-linkers in two-dimensional and three-dimensional cultures, we generate an ECM in which 0-0.92 of total lysine residues are cross-linked. Using an ECM with a range of cross-linking degrees, we demonstrate that the dynamics of invadopodia-related functions have a biphasic relationship to ECM cross-linking. At intermediate levels of ECM cross-linking (0.39), cells exhibit rapid invadopodia protrusion-retraction cycles and rapid calcium spikes, which lead to more frequent MT1-MMP delivery, causing maximal invadopodia-mediated ECM degradation. In contrast, both extremely high or low levels of cross-linking lead to slower invadopodia-related dynamics and lower ECM degradation. Additionally, ß1-integrin inhibition modifies the dynamics of invadopodia-related functions as well as the length of time cells spend in either of the states. Collectively, these data suggest that ß1-integrin-ECM binding nonlinearly translates small physical differences in the extracellular environment to differences in the dynamics of cancer cell behaviors. Understanding the conditions under which invadopodia can be reduced by subtle environment-targeting treatments may lead to combination therapies for preventing metastatic spread.

Multiparametric classification links tumor microenvironments with tumor cell phenotype.

While it has been established that a number of microenvironment components can affect the likelihood of metastasis, the link between microenvironment and tumor cell phenotypes is poorly understood. Here we have examined microenvironment control over two different tumor cell motility phenotypes required for metastasis. By high-resolution multiphoton microscopy of mammary carcinoma in mice, we detected two phenotypes of motile tumor cells, different in locomotion speed. Only slower tumor cells exhibited protrusions with molecular, morphological, and functional characteristics associated with invadopodia. Each region in the primary tumor exhibited either fast- or slow-locomotion. To understand how the tumor microenvironment controls invadopodium formation and tumor cell locomotion, we systematically analyzed components of the microenvironment previously associated with cell invasion and migration. No single microenvironmental property was able to predict the locations of tumor cell phenotypes in the tumor if used in isolation or combined linearly. To solve this, we utilized the support vector machine (SVM) algorithm to classify phenotypes in a nonlinear fashion. This approach identified conditions that promoted either motility phenotype. We then demonstrated that varying one of the conditions may change tumor cell behavior only in a context-dependent manner. In addition, to establish the link between phenotypes and cell fates, we photoconverted and monitored the fate of tumor cells in different microenvironments, finding that only tumor cells in the invadopodium-rich microenvironments degraded extracellular matrix (ECM) and disseminated. The number of invadopodia positively correlated with degradation, while the inhibiting metalloproteases eliminated degradation and lung metastasis, consistent with a direct link among invadopodia, ECM degradation, and metastasis. We have detected and characterized two phenotypes of motile tumor cells in vivo, which occurred in spatially distinct microenvironments of primary tumors. We show how machine-learning analysis can classify heterogeneous microenvironments in vivo to enable prediction of motility phenotypes and tumor cell fate. The ability to predict the locations of tumor cell behavior leading to metastasis in breast cancer models may lead towards understanding the heterogeneity of response to treatment.

N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors.

Invadopodia are proteolytic membrane protrusions formed by highly invasive cancer cells, commonly observed on substrate(s) mimicking extracellular matrix. Although invadopodia are proposed to have roles in cancer invasion and metastasis, direct evidence has not been available. We previously reported that neural Wiskott-Aldrich syndrome protein (N-WASP), a member of WASP family proteins that regulate reorganization of the actin cytoskeleton, is an essential component of invadopodia. Here, we report that N-WASP-mediated invadopodium formation is essential in breast cancer invasion, intravasation and lung metastasis. We established stable cell lines based on MTLn3 rat mammary adenocarcinoma cells that either overexpressed a dominant-negative (DN) N-WASP construct or in which N-WASP expression was silenced by a pSuper N-WASP shRNA. Both the N-WASP shRNA and DN N-WASP cells showed a markedly decreased ability to form invadopodia and degrade extracellular matrix. In addition, formation of invadopodia in primary tumors and collagen I degradation were reduced in the areas of invasion (collagen-rich areas in the invasive edge of the tumor) and in the areas of intravasation (blood-vessel-rich areas). Our results suggest that tumor cells in vivo that have a decreased activity of N-WASP also have a reduced ability to form invadopodia, migrate, invade, intravasate and disseminate to lung compared with tumor cells with parental N-WASP levels.

Retrograde tracing of breast cancer-associated sensory neurons.

Breast cancer is one of the leading causes of mortality among women. The tumor microenvironment, consisting of host cells and extracellular matrix, has been increasingly studied for its interplay with cancer cells, and the resulting effect on tumor progression. While the breast is one of the most innervated organs in the body, the role of neurons, and specifically sensory neurons, has been understudied, mostly for technical reasons. One of the reasons is the anatomy of sensory neurons: sensory neuron somas are located in the spine, and their axons can extend longer than a meter across the body to provide innervation in the breast. Next, neurons are challenging to culture, and there are no cell lines adequately representing the diversity of sensory neurons. Finally, sensory neurons are responsible for transporting several different types of signals to the brain, and there are many different subtypes of sensory neurons. The subtypes of sensory neurons which innervate and interact with breast tumors are unknown. To establish the tools for labeling and subtyping neurons that interact with breast cancer cells, we utilized two retrograde tracer's standards in neuroscience, wheat-germ agglutinin (WGA) and cholera toxin subunit B (CTB). In vitro , we employed primary sensory neurons isolated from mouse dorsal root ganglia, cultured in a custom-built microfluidic device DACIT, that mimics the anatomical compartmentalization of the sensory neuron's soma and axons. In vivo , we utilized both syngeneic and transgenic mouse models of mammary carcinoma. We show that CTB and WGA trace different but overlapping sensory neuronal subpopulations: while WGA is more efficient in labeling CGRP+ neurons, CTB is superior in labeling the NF200+ neurons. Surprisingly, both tracers are also taken up by a significant population of breast cancer cells, both in vitro and in vivo . In summary, we have established methodologies for retrograde tracing of sensory neurons interacting with breast cancer cells. Our tools will be useful for future studies of breast tumor innervation, and development of therapies targeting breast cancer-associated neuron subpopulations of sensory neurons.

DACIT: Device for Axon - Cancer cell Interaction Testing in 2D and 3D.

In this protocol, we describe steps to design, fabricate and use the Device for Axon and Cancer cell Interaction Testing (DACIT) in 2D and in 3D. In the first section, we detail steps to generate the mask, the master and the smooth-on mold. Next, we describe the step-by-step protocol for fabricating the DACIT, loading sensory neurons and cancer cells in 2D or 3D. We compare axonogenesis using PC-12 cell line and primary embryonic or adult sensory neurons, demonstrating the superior neurite growth in primary cells. We demonstrate DACIT can be used to compartmentalize neuronal soma and axons and expose them to different conditions, or to form a temporary gradient of neurotransmitter. Finally, we show that DACIT can be used to measure spheroid invasion in 3D in the presence of axons.

Derivation of human primary prostate epithelial cell lines by differentially targeting the CDKN2A locus along with expression of hTERT.

Prostate cancer (PCa) is the most common cancer diagnosed in men worldwide and was the second leading cause of cancer-related deaths in US males in 2022. Prostate cancer also represents the second highest cancer mortality disparity between non-Hispanic blacks and whites. However, there is a relatively small number of prostate normal and cancer cell lines compared to other cancers. To identify the molecular basis of PCa progression, it is important to have prostate epithelial cell (PrEC) lines as karyotypically normal as possible. Our lab recently developed a novel methodology for the rapid and efficient immortalization of normal human PrEC that combines simultaneous CRISPR-directed inactivation of CDKN2A exon 2 (which directs expression of p16INK4A and p14ARF) and ectopic expression of an hTERT transgene. To optimize this methodology to generate immortalized lines with minimal genetic alterations, we sought to target exon 1α of the CDKN2A locus so that p16INK4A expression is ablated while the exons encoding p14ARF remains unaltered. Here we describe the establishment of two cell lines: one with the above-mentioned p16INK4A only loss, and a second line targeting both products in the CDKN2A locus. We characterize the potential lineage origin of these new cell lines along with our previously obtained clones, revealing distinct gene expression signatures. Based on the analyses of protein markers and RNA expression signatures, these cell lines are most closely related to a subpopulation of basal prostatic cells. Given the simplicity of this one-step methodology and the fact that it uses only the minimal genetic alterations necessary for immortalization, it should also be suitable for the establishment of cell lines from primary prostate tumor samples, an urgent need given the limited number of available prostate cancer cell lines.

Derivation of human primary prostate epithelial cell lines by differentially targeting the CDKN2A locus along with expression of hTERT.

Prostate cancer (PCa) is the most common cancer diagnosed in men worldwide and the second leading cause of cancer-related deaths in US males in 2022. Prostate cancer also represents the second highest cancer mortality disparity between non-Hispanic blacks and whites. However, there is a relatively small number of prostate normal and cancer cell lines compared to other cancers. To identify the molecular basis of PCa progression, it is important to have prostate epithelial cell (PrEC) lines as karyotypically normal as possible. Our lab recently developed a novel methodology for the rapid and efficient immortalization of normal human PrEC that combines simultaneous CRISPR-directed inactivation of CDKN2A exon 2 (which directs expression of p16INK4A and p14ARF) and ectopic expression of an hTERT transgene. To optimize this methodology to generate immortalized lines with minimal genetic alterations, we sought to target exon 1α of the CDKN2A locus so that p16INK4A expression is ablated while p14ARF expression remains unaltered. Here we describe the establishment of two cell lines: one with the above-mentioned p16INK4A only loss, and a second line targeting both products in the CDKN2A locus. We characterize the potential lineage origin of these new cell lines along with our previously obtained clones, revealing distinct gene expression signatures. Based on the analyses of protein markers and RNA expression signatures, these cell lines are most closely related to a subpopulation of basal prostatic cells. Given the simplicity of this one-step methodology and the fact that it uses only the minimal genetic alterations necessary for immortalization, it should also be suitable for the establishment of cell lines from primary prostate tumor samples, an urgent need given the limited number of available prostate cancer cell lines.

Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer.

We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (Mena(INV)) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated Mena(INV) increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by Mena(INV) is dependent on a macrophage-tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of Mena(INV) and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells.

Trogocytosis of cancer-associated fibroblasts promotes pancreatic cancer growth and immune suppression via phospholipid scramblase anoctamin 6 (ANO6).

In pancreatic ductal adenocarcinoma (PDAC), the fibroblastic stroma constitutes most of the tumor mass and is remarkably devoid of functional blood vessels. This raises an unresolved question of how PDAC cells obtain essential metabolites and water-insoluble lipids. We have found a critical role for cancer-associated fibroblasts (CAFs) in obtaining and transferring lipids from blood-borne particles to PDAC cells via trogocytosis of CAF plasma membranes. We have also determined that CAF-expressed phospholipid scramblase anoctamin 6 (ANO6) is an essential CAF trogocytosis regulator required to promote PDAC cell survival. During trogocytosis, cancer cells and CAFs form synapse-like plasma membranes contacts that induce cytosolic calcium influx in CAFs via Orai channels. This influx activates ANO6 and results in phosphatidylserine exposure on CAF plasma membrane initiating trogocytosis and transfer of membrane lipids, including cholesterol, to PDAC cells. Importantly, ANO6-dependent trogocytosis also supports the immunosuppressive function of pancreatic CAFs towards cytotoxic T cells by promoting transfer of excessive amounts of cholesterol. Further, blockade of ANO6 antagonizes tumor growth via disruption of delivery of exogenous cholesterol to cancer cells and reverses immune suppression suggesting a potential new strategy for PDAC therapy.

Invadopodia enable cooperative invasion and metastasis of breast cancer cells.

Invasive and non-invasive cancer cells can invade together during cooperative invasion. However, the events leading to it, role of the epithelial-mesenchymal transition and the consequences this may have on metastasis are unknown. In this study, we demonstrate that the isogenic 4T1 and 67NR breast cancer cells sort from each other in 3D spheroids, followed by cooperative invasion. By time-lapse microscopy, we show that the invasive 4T1 cells move more persistently compared to non-invasive 67NR, sorting and accumulating at the spheroid-matrix interface, a process dependent on cell-matrix adhesions and independent from E-cadherin cell-cell adhesions. Elimination of invadopodia in 4T1 cells blocks invasion, demonstrating that invadopodia requirement is limited to leader cells. Importantly, we demonstrate that cells with and without invadopodia can also engage in cooperative metastasis in preclinical mouse models. Altogether, our results suggest that a small number of cells with invadopodia can drive the metastasis of heterogeneous cell clusters.

Intravital imaging of metastatic behavior through a mammary imaging window.

We report a technique to evaluate the same tumor microenvironment over multiple intravital imaging sessions in living mice. We optically marked individual tumor cells expressing photoswitchable proteins in an orthotopic mammary carcinoma and followed them for extended periods through a mammary imaging window. We found that two distinct microenvironments in the same orthotopic mammary tumor affected differently the invasion and intravasation of tumor cells.

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users' needs.

Pharmacodynamic Studies of Fluorescent Diamond Carriers of Doxorubicin in Liver Cancer Cells and Colorectal Cancer Organoids.

BACKGROUND: We recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs. PURPOSE: To generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer. METHODS: FDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions. RESULTS: FDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence. CONCLUSION: The rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.

Frontiers in Intravital Multiphoton Microscopy of Cancer.

Background: Cancer is a highly complex disease which involves the co-operation of tumor cells with multiple types of host cells and the extracellular matrix. Cancer studies which rely solely on static measurements of individual cell types are insufficient to dissect this complexity. In the last two decades, intravital microscopy has established itself as a powerful technique that can significantly improve our understanding of cancer by revealing the dynamic interactions governing cancer initiation, progression and treatment effects, in living animals. This review focuses on intravital multiphoton microscopy (IV-MPM) applications in mouse models of cancer. Recent Findings: IV-MPM studies have already enabled a deeper understanding of the complex events occurring in cancer, at the molecular, cellular and tissue levels. Multiple cells types, present in different tissues, influence cancer cell behavior via activation of distinct signaling pathways. As a result, the boundaries in the field of IV-MPM are continuously being pushed to provide an integrated comprehension of cancer. We propose that optics, informatics and cancer (cell) biology are co-evolving as a new field. We have identified four emerging themes in this new field. First, new microscopy systems and image processing algorithms are enabling the simultaneous identification of multiple interactions between the tumor cells and the components of the tumor microenvironment. Second, techniques from molecular biology are being exploited to visualize subcellular structures and protein activities within individual cells of interest, and relate those to phenotypic decisions, opening the door for "in vivo cell biology". Third, combining IV-MPM with additional imaging modalities, or omics studies, holds promise for linking the cell phenotype to its genotype, metabolic state or tissue location. Finally, the clinical use of IV-MPM for analyzing efficacy of anti-cancer treatments is steadily growing, suggesting a future role of IV-MPM for personalized medicine. Conclusion: IV-MPM has revolutionized visualization of tumor-microenvironment interactions in real time. Moving forward, incorporation of novel optics, automated image processing, and omics technologies, in the study of cancer biology, will not only advance our understanding of the underlying complexities but will also leverage the unique aspects of IV-MPM for clinical use.

Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy.

We previously customized a Nipkow spinning disk confocal microscope (SDCM) to acquire 4D data for live, intraerythrocytic malarial parasites [Gligorijevic B, McAllister R, Urbach JS, Roepe, PD. Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria. Biochemistry 2006;45:12400-10]. We reported that chloroquine (CQ) treatment did not appear to affect progress through the cell cycle, and suggested that toxicity may be manifested post-schizogony. We now use SDCM, synchronized cell culture and continuous vs. bolus drug dosing to investigate stage specific CQ effects in detail. We develop a novel, extremely rapid method for counting schizont nuclei in 3D. We then quantify schizont nuclei and hemozoin (Hz) production for live parasite cultures pulsed with CQ at different stages in the cell cycle and find that bolus treatment of rings affects the multiplicity of nuclear division. We quantify parasitemia and merozoite development in subsequent cycles following bolus CQ exposure and find that a portion of CQ toxicity is manifested post-schizogony as "delayed death". Using these methods and others we compare CQ sensitive (CQS) vs. resistant (CQR) strains as well as transfectants that are CQR via introduction of mutant PfCRT. Surprisingly, we find that PfCRT confers resistance to CQ administered at the very early ring stage of development, wherein a digestive vacuole is not yet formed, as well as at the schizont stage, wherein Hz production is thought to plateau. Taken together, these data force a rethinking of CQ pharmacology and the mechanism of CQR.

Intravital Imaging of Tumor Cell Motility in the Tumor Microenvironment Context.

Cancer cell motility and invasion are key features of metastatic tumors. Both are highly linked to tumor microenvironmental parameters, such as collagen architecture or macrophage density. However, due to the genetic, epigenetic and microenvironmental heterogeneities, only a small portion of tumor cells in the primary tumor are motile and furthermore, only a small portion of those will metastasize. This creates a challenge in predicting metastatic fate of single cells based on the phenotype they exhibit in the primary tumor. To overcome this challenge, tumor cell subpopulations need to be monitored at several timescales, mapping their phenotype in primary tumor as well as their potential homing to the secondary tumor site. Additionally, to address the spatial heterogeneity of the tumor microenvironment and how it relates to tumor cell phenotypes, large numbers of images need to be obtained from the same tumor. Finally, as the microenvironment complexity results in nonlinear relationships between tumor cell phenotype and its surroundings, advanced statistical models are required to interpret the imaging data. Toward improving our understanding of the relationship between cancer cell motility, the tumor microenvironment context and successful metastasis, we have developed several intravital approaches for continuous and longitudinal imaging, as well as data classification via support vector machine (SVM) algorithm. We also describe methods that extend the capabilities of intravital imaging by postsacrificial microscopy of the lung as well as correlative immunofluorescence in the primary tumor.

Contact guidance is cell cycle-dependent.

Cancer cell migration is essential for metastasis, during which cancer cells move through the tumor and reach the blood vessels. In vivo, cancer cells are exposed to contact guidance and chemotactic cues. Depending on the strength of such cues, cells will migrate in a random or directed manner. While similar cues may also stimulate cell proliferation, it is not clear whether cell cycle progression affects migration of cancer cells and whether this effect is different in random versus directed migration. In this study, we tested the effect of cell cycle progression on contact guided migration in 2D and 3D environments, in the breast carcinoma cell line, FUCCI-MDA-MB-231. The results were quantified from live cell microscopy images using the open source lineage editing and validation image analysis tools (LEVER). In 2D, cells were placed inside 10 µm-wide microchannels to stimulate contact guidance, with or without an additional chemotactic gradient of the soluble epidermal growth factor. In 3D, contact guidance was modeled by aligned collagen fibers. In both 2D and 3D, contact guidance was cell cycle-dependent, while the addition of the chemo-attractant gradient in 2D increased cell velocity and persistence in directionally migrating cells, regardless of their cell cycle phases. In both 2D and 3D contact guidance, cells in the G1 phase of the cell cycle outperformed cells in the S/G2 phase in terms of migration persistence and instantaneous velocity. These data suggest that in the presence of contact guidance cues in vivo, breast carcinoma cells in the G1 phase of the cell cycle may be more efficient in reaching the neighboring vasculature.