Cellular interactions in tumor microenvironment during breast cancer progression: new frontiers and implications for novel therapeutics.

The breast cancer tumor microenvironment (TME) is dynamic, with various immune and non-immune cells interacting to regulate tumor progression and anti-tumor immunity. It is now evident that the cells within the TME significantly contribute to breast cancer progression and resistance to various conventional and newly developed anti-tumor therapies. Both immune and non-immune cells in the TME play critical roles in tumor onset, uncontrolled proliferation, metastasis, immune evasion, and resistance to anti-tumor therapies. Consequently, molecular and cellular components of breast TME have emerged as promising therapeutic targets for developing novel treatments. The breast TME primarily comprises cancer cells, stromal cells, vasculature, and infiltrating immune cells. Currently, numerous clinical trials targeting specific TME components of breast cancer are underway. However, the complexity of the TME and its impact on the evasion of anti-tumor immunity necessitate further research to develop novel and improved breast cancer therapies. The multifaceted nature of breast TME cells arises from their phenotypic and functional plasticity, which endows them with both pro and anti-tumor roles during tumor progression. In this review, we discuss current understanding and recent advances in the pro and anti-tumoral functions of TME cells and their implications for developing safe and effective therapies to control breast cancer progress.

The impact of tumor microenvironment: unraveling the role of physical cues in breast cancer progression.

Metastasis accounts for the vast majority of breast cancer-related fatalities. Although the contribution of genetic and epigenetic modifications to breast cancer progression has been widely acknowledged, emerging evidence underscores the pivotal role of physical stimuli in driving breast cancer metastasis. In this review, we summarize the changes in the mechanics of the breast cancer microenvironment and describe the various forces that impact migrating and circulating tumor cells throughout the metastatic process. We also discuss the mechanosensing and mechanotransducing molecules responsible for promoting the malignant phenotype in breast cancer cells. Gaining a comprehensive understanding of the mechanobiology of breast cancer carries substantial potential to propel progress in prognosis, diagnosis, and patient treatment.

Cytoplasmic accumulation and plasma membrane association of anillin and Ect2 promote confined migration and invasion.

Cells migrating in confinement experience mechanical challenges whose consequences on cell migration machinery remain only partially understood. Here, we demonstrate that a pool of the cytokinesis regulatory protein anillin is retained during interphase in the cytoplasm of different cell types. Confinement induces recruitment of cytoplasmic anillin to plasma membrane at the poles of migrating cells, which is further enhanced upon nuclear envelope (NE) rupture(s). Rupture events also enable the cytoplasmic egress of predominantly nuclear RhoGEF Ect2. Anillin and Ect2 redistributions scale with microenvironmental stiffness and confinement, and are observed in confined cells in vitro and in invading tumor cells in vivo. Anillin, which binds actomyosin at the cell poles, and Ect2, which activates RhoA, cooperate additively to promote myosin II contractility, and promote efficient invasion and extravasation. Overall, our work provides a mechanistic understanding of how cytokinesis regulators mediate RhoA/ROCK/myosin II-dependent mechanoadaptation during confined migration and invasive cancer progression.

Coordinated in confined migration: crosstalk between the nucleus and ion channel-mediated mechanosensation.

Cell surface and intracellular mechanosensors enable cells to perceive different geometric, topographical, and physical cues. Mechanosensitive ion channels (MICs) localized at the cell surface and on the nuclear envelope (NE) are among the first to sense and transduce these signals. Beyond compartmentalizing the genome of the cell and its transcription, the nucleus also serves as a mechanical gauge of different physical and topographical features of the tissue microenvironment. In this review, we delve into the intricate mechanisms by which the nucleus and different ion channels regulate cell migration in confinement. We review evidence suggesting an interplay between macromolecular nuclear-cytoplasmic transport (NCT) and ionic transport across the cell membrane during confined migration. We also discuss the roles of the nucleus and ion channel-mediated mechanosensation, whether acting independently or in tandem, in orchestrating migratory mechanoresponses. Understanding nuclear and ion channel sensing, and their crosstalk, is critical to advancing our knowledge of cell migration in health and disease.

Metronomic Administration of Topotecan Alone and in Combination with Docetaxel Inhibits Epithelial-mesenchymal Transition in Aggressive Variant Prostate Cancers.

Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiotherapy remain the primary treatment for patients with early-stage prostate cancer (castration-sensitive prostate cancer). Following ADT, many patients ultimately develop metastatic castration-resistant prostate cancer (mCRPC). Standard chemotherapy options for CRPC are docetaxel (DTX) and cabazitaxel, which increase median survival, although the development of resistance is common. Cancer stem-like cells possess mesenchymal phenotypes [epithelial-to-mesenchymal transition (EMT)] and play crucial roles in tumor initiation and progression of mCRPC. We have shown that low-dose continuous administration of topotecan (METRO-TOPO) inhibits prostate cancer growth by interfering with key cancer pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis [(RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq)], and we observed greater expression of several EMT markers, including Vimentin, hyaluronan synthase-3, S100 calcium binding protein A6, TGFB1, CD44, CD55, and CD109 in European American and African American aggressive variant prostate cancer (AVPC) subtypes-mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell subclonal populations in mCRPC. Furthermore, metronomic-topotecan single agent and combinations with DTX downregulated these EMT markers as well as CD44+ and CD44+/CD133+ "stem-like" cell populations. A microfluidic chip-based cell invasion assay revealed that METRO-TOPO treatment as a single agent or in combination with DTX was potentially effective against invasive prostate cancer spread. Our RNA-seq and scRNA-seq analysis were supported by in silico and in vitro studies, suggesting METRO-TOPO combined with DTX may inhibit oncogenic progression by reducing cancer stemness in AVPC through the inhibition of EMT markers and multiple oncogenic factors/pathways. Significance: The utilization of metronomic-like dosing regimens of topotecan alone and in combination with DTX resulted in the suppression of makers associated with EMT and stem-like cell populations in AVPC models. The identification of molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression were achieved using bulk RNA-seq and single-cell-omics methodologies.

Downregulation of YAP Activity Restricts P53 Hyperactivation to Promote Cell Survival in Confinement.

Cell migration through confining three dimensional (3D) topographies can lead to loss of nuclear envelope integrity, DNA damage, and genomic instability. Despite these detrimental phenomena, cells transiently exposed to confinement do not usually die. Whether this is also true for cells subjected to long-term confinement remains unclear at present. To investigate this, photopatterning and microfluidics are employed to fabricate a high-throughput device that circumvents limitations of previous cell confinement models and enables prolonged culture of single cells in microchannels with physiologically relevant length scales. The results of this study show that continuous exposure to tight confinement can trigger frequent nuclear envelope rupture events, which in turn promote P53 activation and cell apoptosis. Migrating cells eventually adapt to confinement and evade cell death by downregulating YAP activity. Reduced YAP activity, which is the consequence of confinement-induced YAP1/2 translocation to the cytoplasm, suppresses the incidence of nuclear envelope rupture and abolishes P53-mediated cell death. Cumulatively, this work establishes advanced, high-throughput biomimetic models for better understanding cell behavior in health and disease, and underscores the critical role of topographical cues and mechanotransduction pathways in the regulation of cell life and death.

Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement.

Cells tune adherens junction dynamics to regulate epithelial integrity in diverse (patho)physiological processes, including cancer metastasis. We hypothesized that the spatially confining architecture of peritumor stroma promotes metastatic cell dissemination by remodeling cell-cell adhesive interactions. By combining microfluidics with live-cell imaging, FLIM/FRET biosensors, and optogenetic tools, we show that confinement induces leader cell dissociation from cohesive ensembles. Cell dissociation is triggered by myosin IIA (MIIA) dismantling of E-cadherin cell-cell junctions, as recapitulated by a mathematical model. Elevated MIIA contractility is controlled by RhoA/ROCK activation, which requires distinct guanine nucleotide exchange factors (GEFs). Confinement activates RhoA via nucleocytoplasmic shuttling of the cytokinesis-regulatory proteins RacGAP1 and Ect2 and increased microtubule dynamics, which results in the release of active GEF-H1. Thus, confining microenvironments are sufficient to induce cell dissemination from primary tumors by remodeling E-cadherin cell junctions via the interplay of microtubules, nuclear trafficking, and RhoA/ROCK/MIIA pathway and not by down-regulating E-cadherin expression.

Integrating Pharmacogenomics Data-Driven Computational Drug Prediction with Single-Cell RNAseq to Demonstrate the Efficacy of a NAMPT Inhibitor against Aggressive, Taxane-Resistant, and Stem-like Cells in Lethal Prostate Cancer.

Metastatic prostate cancer/PCa is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and, therefore, androgen deprivation therapies/ADT-sensitive. However, eventual resistance to standard medical castration (AR-inhibitors) and secondary chemotherapies (taxanes) is nearly universal. Further, the presence of cancer stem-like cells (EMT/epithelial-to-mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/lethal/advanced variants of PCa (AVPC). In this study, we introduced a pharmacogenomics data-driven optimization-regularization-based computational prediction algorithm ("secDrugs") to predict novel drugs against lethal PCa. Integrating secDrug with single-cell RNA-sequencing/scRNAseq as a 'Double-Hit' drug screening tool, we demonstrated that single-cells representing drug-resistant and stem-cell-like cells showed high expression of the NAMPT pathway genes, indicating potential efficacy of the secDrug FK866 which targets NAMPT. Next, using several cell-based assays, we showed substantial impact of FK866 on clinically advanced PCa as a single agent and in combination with taxanes or AR-inhibitors. Bulk-RNAseq and scRNAseq revealed that, in addition to NAMPT inhibition, FK866 regulates tumor metastasis, cell migration, invasion, DNA repair machinery, redox homeostasis, autophagy, as well as cancer stemness-related genes, HES1 and CD44. Further, we combined a microfluidic chip-based cell migration assay with a traditional cell migration/'scratch' assay and demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using PCa patient datasets, we showed that FK866 is potentially capable of reversing the expression of several genes associated with biochemical recurrence, including IFITM3 and LTB4R. Thus, using FK866 as a proof-of-concept candidate for drug repurposing, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.

Extracellular fluid viscosity enhances cell migration and cancer dissemination.

Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology.

Polarized NHE1 and SWELL1 regulate migration direction, efficiency and metastasis.

Cell migration regulates diverse (patho)physiological processes, including cancer metastasis. According to the Osmotic Engine Model, polarization of NHE1 at the leading edge of confined cells facilitates water uptake, cell protrusion and motility. The physiological relevance of the Osmotic Engine Model and the identity of molecules mediating cell rear shrinkage remain elusive. Here, we demonstrate that NHE1 and SWELL1 preferentially polarize at the cell leading and trailing edges, respectively, mediate cell volume regulation, cell dissemination from spheroids and confined migration. SWELL1 polarization confers migration direction and efficiency, as predicted mathematically and determined experimentally via optogenetic spatiotemporal regulation. Optogenetic RhoA activation at the cell front triggers SWELL1 re-distribution and migration direction reversal in SWELL1-expressing, but not SWELL1-knockdown, cells. Efficient cell reversal also requires Cdc42, which controls NHE1 repolarization. Dual NHE1/SWELL1 knockdown inhibits breast cancer cell extravasation and metastasis in vivo, thereby illustrating the physiological significance of the Osmotic Engine Model.

Migration and 3D Traction Force Measurements inside Compliant Microchannels.

Cells migrate in vivo through channel-like tracks. While polydimethylsiloxane devices emulate such tracks in vitro, their channel walls are impermeable and have supraphysiological stiffness. Existing hydrogel-based platforms address these issues but cannot provide high-throughput analysis of cell motility in independently controllable stiffness and confinement. We herein develop polyacrylamide (PA)-based microchannels of physiological stiffness and prescribed dimensions for high-throughput analysis of cell migration and identify a biphasic dependence of speed upon confinement and stiffness. By utilizing novel four-walled microchannels with heterogeneous stiffness, we reveal the distinct contributions of apicolateral versus basal microchannel wall stiffness to confined versus unconfined migration. While the basal wall stiffness dictates unconfined migration, apicolateral stiffness controls confined migration. By tracking nanobeads embedded within channel walls, we innovate three-dimensional traction force measurements around spatially confining cells at subcellular resolution. Our unique and highly customizable device fabrication strategy provides a physiologically relevant in vitro platform to study confined cells.

Kidney epithelial cells are active mechano-biological fluid pumps.

The role of mechanical forces driving kidney epithelial fluid transport and morphogenesis in kidney diseases is unclear. Here, using a microfluidic platform to recapitulate fluid transport activity of kidney cells, we report that renal epithelial cells can actively generate hydraulic pressure gradients across the epithelium. The fluidic flux declines with increasing hydraulic pressure until a stall pressure, in a manner similar to mechanical fluid pumps. For normal human kidney cells, the fluidic flux is from apical to basal, and the pressure is higher on the basal side. For human Autosomal Dominant Polycystic Kidney Disease cells, the fluidic flux is reversed from basal to apical. Molecular and proteomic studies reveal that renal epithelial cells are sensitive to hydraulic pressure gradients, changing gene expression profiles and spatial arrangements of ion exchangers and the cytoskeleton in different pressure conditions. These results implicate mechanical force and hydraulic pressure as important variables during kidney function and morphological change, and provide insights into pathophysiological mechanisms underlying the development and transduction of hydraulic pressure gradients.

The fluid shear stress sensor TRPM7 regulates tumor cell intravasation.

Tumor cell intravasation preferentially occurs in regions of low fluid shear because high shear is detrimental to tumor cells. Here, we describe a molecular mechanism by which cells avoid high shear during intravasation. The transition from migration to intravasation was modeled using a microfluidic device where cells migrating inside longitudinal tissue-like microchannels encounter an orthogonal channel in which fluid flow induces physiological shear stresses. This approach was complemented with intravital microscopy, patch-clamp, and signal transduction imaging techniques. Fluid shear-induced activation of the transient receptor potential melastatin 7 (TRPM7) channel promotes extracellular calcium influx, which then activates RhoA/myosin-II and calmodulin/IQGAP1/Cdc42 pathways to coordinate reversal of migration direction, thereby avoiding shear stress. Cells displaying higher shear sensitivity due to higher TRPM7 activity levels intravasate less efficiently and establish less invasive metastatic lesions. This study provides a mechanistic interpretation for the role of shear stress and its sensor, TRPM7, in tumor cell intravasation.

A Procarcinogenic Colon Microbe Promotes Breast Tumorigenesis and Metastatic Progression and Concomitantly Activates Notch and ß-Catenin Axes.

The existence of distinct breast microbiota has been recently established, but their biological impact in breast cancer remains elusive. Focusing on the shift in microbial community composition in diseased breast compared with normal breast, we identified the presence of Bacteroides fragilis in cancerous breast. Mammary gland as well as gut colonization with enterotoxigenic Bacteroides fragilis (ETBF), which secretes B. fragilis toxin (BFT), rapidly induces epithelial hyperplasia in the mammary gland. Breast cancer cells exposed to BFT exhibit "BFT memory" from the initial exposure. Intriguingly, gut or breast duct colonization with ETBF strongly induces growth and metastatic progression of tumor cells implanted in mammary ducts, in contrast to nontoxigenic Bacteroides fragilis. This work sheds light on the oncogenic impact of a procarcinogenic colon bacterium ETBF on breast cancer progression, implicates the ß-catenin and Notch1 axis as its functional mediators, and proposes the concept of "BFT memory" that can have far-reaching biological implications after initial exposure to ETBF. SIGNIFICANCE: B. fragilis is an inhabitant of breast tissue, and gut or mammary duct colonization with ETBF triggers epithelial hyperplasia and augments breast cancer growth and metastasis. Short-term exposure to BFT elicits a "BFT memory" with long-term implications, functionally mediated by the ß-catenin and Notch1 pathways.This article is highlighted in the In This Issue feature, p. 995.

Myosin 10 Regulates Invasion, Mitosis, and Metabolic Signaling in Glioblastoma.

Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other, implying that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice can survive to adulthood, implying that normal cells can compensate for its loss; its deletion impairs invasion, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is active against glioblastoma by itself, synergizes with other clinically available therapeutics, may have acceptable side effects in normal tissues, and has potential as a heretofore unexplored therapeutic approach for this disease.

Dorsoventral polarity directs cell responses to migration track geometries.

How migrating cells differentially adapt and respond to extracellular track geometries remains unknown. Using intravital imaging, we demonstrate that invading cells exhibit dorsoventral (top-to-bottom) polarity in vivo. To investigate the impact of dorsoventral polarity on cell locomotion through different confining geometries, we fabricated microchannels of fixed cross-sectional area, albeit with distinct aspect ratios. Vertical confinement, exerted along the dorsoventral polarity axis, induces myosin II-dependent nuclear stiffening, which results in RhoA hyperactivation at the cell poles and slow bleb-based migration. In lateral confinement, directed perpendicularly to the dorsoventral polarity axis, the absence of perinuclear myosin II fails to increase nuclear stiffness. Hence, cells maintain basal RhoA activity and display faster mesenchymal migration. In summary, by integrating microfabrication, imaging techniques, and intravital microscopy, we demonstrate that dorsoventral polarity, observed in vivo and in vitro, directs cell responses in confinement by spatially tuning RhoA activity, which controls bleb-based versus mesenchymal migration.

Confinement hinders motility by inducing RhoA-mediated nuclear influx, volume expansion, and blebbing.

Cells migrate in vivo through complex confining microenvironments, which induce significant nuclear deformation that may lead to nuclear blebbing and nuclear envelope rupture. While actomyosin contractility has been implicated in regulating nuclear envelope integrity, the exact mechanism remains unknown. Here, we argue that confinement-induced activation of RhoA/myosin-II contractility, coupled with LINC complex-dependent nuclear anchoring at the cell posterior, locally increases cytoplasmic pressure and promotes passive influx of cytoplasmic constituents into the nucleus without altering nuclear efflux. Elevated nuclear influx is accompanied by nuclear volume expansion, blebbing, and rupture, ultimately resulting in reduced cell motility. Moreover, inhibition of nuclear efflux is sufficient to increase nuclear volume and blebbing on two-dimensional surfaces, and acts synergistically with RhoA/myosin-II contractility to further augment blebbing in confinement. Cumulatively, confinement regulates nuclear size, nuclear integrity, and cell motility by perturbing nuclear flux homeostasis via a RhoA-dependent pathway.

Restoring extracellular matrix synthesis in senescent stem cells.

Collagen type III (COL3) is one of the 3 major collagens in the body, and loss of expression or mutations in the COL3 gene have been associated with the onset of vascular diseases such the Ehlers-Danlos syndrome. Previous work reported a significant reduction of COL3 in tissues such as skin and vessels with aging. In agreement, we found that COL3 was significantly reduced in senescent human mesenchymal stem cells and myofibroblasts derived from patients with Hutchinson-Gilford progeria syndrome, a premature aging syndrome. Most notably, we discovered that ectopic expression of the embryonic transcription factor Nanog homeobox (NANOG) restored COL3 expression by restoring the activity of the TGF-ß pathway that was impaired in senescent cells. RNA sequencing analysis showed that genes associated with the activation of the TGF-ß pathway were up-regulated, whereas negative regulators of the pathway were down-regulated upon NANOG expression. Chromatin immunoprecipitation sequencing and immunoprecipitation experiments revealed that NANOG bound to the mothers against decapentaplegic (SMAD)2 and SMAD3 promoters, in agreement with increased expression and phosphorylation levels of both proteins. Using chemical inhibition, short hairpin RNA knockdown, and gain of function approaches, we established that both SMAD2 and SMAD3 were necessary to mediate the effects of NANOG, but SMAD3 overexpression was also sufficient for COL3 production. In summary, NANOG restored production of COL3, which was impaired by cellular aging, suggesting novel strategies to restore the impaired extracellular matrix production and biomechanical function of aged tissues, with potential implications for regenerative medicine and anti-aging treatments.-Rong, N., Mistriotis, P., Wang, X., Tseropoulos, G., Rajabian, N., Zhang, Y., Wang, J., Liu, S., Andreadis, S. T. Restoring extracellular matrix synthesis in senescent stem cells.

Cell sensing and decision-making in confinement: The role of TRPM7 in a tug of war between hydraulic pressure and cross-sectional area.

How cells sense hydraulic pressure and make directional choices in confinement remains elusive. Using trifurcating Ψ-like microchannels of different hydraulic resistances and cross-sectional areas, we discovered that the TRPM7 ion channel is the critical mechanosensor, which directs decision-making of blebbing cells toward channels of lower hydraulic resistance irrespective of their cross-sectional areas. Hydraulic pressure-mediated TRPM7 activation triggers calcium influx and supports a thicker cortical actin meshwork containing an elevated density of myosin-IIA. Cortical actomyosin shields cells against external forces and preferentially directs cell entrance in low resistance channels. Inhibition of TRPM7 function or actomyosin contractility renders cells unable to sense different resistances and alters the decision-making pattern to cross-sectional area-based partition. Cell distribution in microchannels is captured by a mathematical model based on the maximum entropy principle using cortical actin as a key variable. This study demonstrates the unique role of TRPM7 in controlling decision-making and navigating migration in complex microenvironments.

A microfluidic assay for the quantification of the metastatic propensity of breast cancer specimens.

The challenge of predicting which patients with breast cancer will develop metastases leads to the overtreatment of patients with benign disease and to the inadequate treatment of aggressive cancers. Here, we report the development and testing of a microfluidic assay that quantifies the abundance and proliferative index of migratory cells in breast cancer specimens, for the assessment of their metastatic propensity and for the rapid screening of potential antimetastatic therapeutics. On the basis of the key roles of cell motility and proliferation in cancer metastasis, the device accurately predicts the metastatic potential of breast cancer cell lines and of patient-derived xenografts. Compared with unsorted cancer cells, highly motile cells isolated by the device exhibited similar tumourigenic potential but markedly increased metastatic propensity in vivo. RNA sequencing of the highly motile cells revealed an enrichment of motility-related and survival-related genes. The approach might be developed into a companion assay for the prediction of metastasis in patients and for the selection of effective therapeutic regimens.