AXL expression reflects tumor-immune cell dynamics impacting outcome in non-small cell lung cancer patients treated with immune checkpoint inhibitor monotherapy.

Introduction: AXL receptor expression is proposed to confer immune-checkpoint inhibitor (ICI)-resistance in non-small cell lung cancer (NSCLC) patients. We sought to interrogate AXL expression in conjunction with mutational and tumor-microenvironmental features to uncover predictive mechanisms of resistance in ICI-treated NSCLC patients. Methods: Tumor samples from 111 NSCLC patients treated with ICI-monotherapy were analyzed by immunohistochemistry for tumor- and immune-AXL expression. Subsets of patients were analyzed by whole-exome sequencing (n = 44) and imaging mass cytometry (n = 14). Results were related to ICI-outcome measurements. Results: Tumor-cell AXL expression correlated with aggressive phenotypic features including reduced OS in patients treated with ICIs (P = 0.04) after chemotherapy progression, but conversely associated with improved disease control (P = 0.045) in ICI-treated, PD-L1 high first-line patients. AXL+ immune-cell infiltration correlated with total immune-cell infiltration and improved overall outcomes (PFS: P = 0.044, OS: P = 0.054). Tumor-cell AXL-upregulation showed enrichment in mutations associated with PD-L1-upregulation and ICI-response such as MUC4 and ZNF469, as well as adverse mutations including CSMD1 and LRP1B which associated with an immune-suppressed tumor phenotype and poor ICI prognosis particularly within chemotherapy-treated patients. Tumor mutational burden had no effect on ICI-outcomes and was associated with a lack of tumor-infiltrating immune cells. Spatial-immunophenotyping provided evidence that tumor-cell AXL-upregulation and adverse mutations modulate the tumor microenvironment in favor of infiltrating, activated neutrophils over anti-tumor immune-subsets including CD4 and CD8 T-cells. Conclusion: Tumor-cell AXL-upregulation correlated with distinct oncotypes and microenvironmental immune-profiles that define chemotherapy-induced mechanisms of ICI-resistance, which suggests the combination of AXL inhibitors with current chemoimmunotherapy regimens can benefit NSCLC patients.

Increased tumor cell expression of Axl is a marker of aggressive features in breast cancer among African women.

Axl, a receptor tyrosine kinase belonging to the Tyro/Axl/Mer (TAM) family, has been shown to be overexpressed in breast cancer with poor outcome. Moreover, Axl was associated with a basal-like phenotype (BLP) in these tumors. Our aim was to investigate Axl expression in breast cancers from an African population since these tumors are known to be aggressive and have a high frequency of the basal-like phenotype. We studied 170 paraffin-embedded breast carcinoma cases by tissue microarrays and immunohistochemical methods. In total, 128 tumor cases (75%) had strong Axl expression and 42 cases (25%) had weak or negative staining. Strong expression of Axl was associated with high tumor grade (p < 0.0005), estrogen receptor (ER) negativity (p = 0.024), p53 expression (p = 0.004), P-cadherin positivity (p = 0.017), and basal-like phenotypic profiles BLP2 (p = 0.033) and BLP3 (p = 0.022). In addition, Axl overexpression also showed an association with markers of tumor cell proliferation and tumor angiogenesis. In conclusion, our findings indicate strong expression of Axl in a high proportion of breast cancer cases among African women and associations with markers of aggressive features, indicating poor prognosis. These findings suggest Axl as a potential therapeutic target in this population.

Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets.

BACKGROUND: The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels. RESULTS: In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells. CONCLUSION: The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.

Contextual compound screening for improved therapeutic discovery.

Cellular behaviors are governed by combinations of systemic and microenvironmental factors; together, these regulate cell signaling responses to growth factors. This contextual microenvironmental influence also determines drug sensitivity. Hence using in vitro systems that model contextual cellular behavior is highly beneficial for effective therapeutic development. Angiogenesis (formation of blood vessels) is driven by a series of dynamic endothelial cell signaling responses to growth factors under the influence of the vascular extracellular matrix and adjacent pericytes. In vitro primary human vascular cell co-cultures self-assemble into capillary-like structures through reciprocal heterotypic interactions that mimic angiogenic context dynamics. By using temporal live-cell imaging-based analysis, unique angiogenic microenvironments can be delineated to quantify the contextual activity of compound inhibitors. We used this in vitro organotypic contextual screening approach to conduct structure-activity relationship analysis on a combretastatin A-4 analogue series to identify novel compounds with potent vascular disrupting activity in vivo.

p63 attenuates epithelial to mesenchymal potential in an experimental prostate cell model.

The transcription factor p63 is central for epithelial homeostasis and development. In our model of epithelial to mesenchymal transition (EMT) in human prostate cells, p63 was one of the most down-regulated transcription factors during EMT. We therefore investigated the role of p63 in EMT. Over-expression of the predominant epithelial isoform ΔNp63α in mesenchymal type cells of the model led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). This was corroborated by a reciprocal effect when p63 was knocked down in epithelial EP156T cells. Global gene expression analyses showed that ΔNp63α induced gene modules involved in both cell-to-cell and cell-to-extracellular-matrix junctions in mesenchymal type cells. Genome-wide analysis of p63 binding sites using ChIP-seq analyses confirmed binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells. DH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knock-down of these factors, respectively, were not sufficient alone or in combination with ΔNp63α to reverse completely the mesenchymal phenotype. The partial reversion of epithelial to mesenchymal transition might reflect the ability of ΔNp63α, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The utility of ΔNp63α expression and the potential of reduced EMP in order to counteract metastasis warrant further investigation.

Expression of TP53 isoforms p53ß or p53γ enhances chemosensitivity in TP53(null) cell lines.

The carboxy-terminal truncated p53 alternative spliced isoforms, p53ß and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53(null) background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53ß and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53ß and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1)), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53ß protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53ß and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53ß and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.

Nitroreductase, a near-infrared reporter platform for in vivo time-domain optical imaging of metastatic cancer.

The ability to visualize reporter gene expression in vivo has revolutionized all facets of biologic investigation and none more so than imaging applications in oncology. Near-infrared reporter gene imaging may facilitate more accurate evaluation of chemotherapeutic response in preclinical models of orthotopic and metastatic cancers. We report the development of a cell permeable, quenched squarine probe (CytoCy5S), which is reduced by Escherichia coli nitroreductase (NTR), resulting in a near-infrared fluorescent product. Time-domain molecular imaging of NTR/CytoCy5S reporter platform permitted noninvasive monitoring of disease progression in orthotopic xenografts of disseminated leukemia, lung, and metastatic breast cancer. This methodology facilitated therapeutic evaluation of NTR gene-directed enzymatic prodrug therapy with conventional metronidazole antibiotics. These studies show NTR/CytoCy5S as a near-infrared gene reporter system with broad preclinical and prospective clinical applications within imaging, and gene therapy, of cancer.

Efficient in vivo vascularization of tissue-engineering scaffolds.

The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and perivascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial and vascular smooth muscle cells were seeded at different ratios in poly-L-lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue-engineering context was strongly enhanced in implants seeded with a complete complement of blood vessel components: human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3D image reconstruction analysis of human pulmonary artery smooth muscle cell (hPASMC) distribution within vascularized implants was non-random and displayed a preferential perivascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.

Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival.

Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.

A novel imaging-based high-throughput screening approach to anti-angiogenic drug discovery.

The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, high-throughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network of tubular endothelial structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGF-switch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot high-throughput screening campaign was used to define informative imaging parameters and develop a follow-up dose-response scheme for hit characterization. High-throughput screening using the EC-vSMC co-culture assay establishes a new platform to screen for novel anti-angiogenic compounds for cancer therapy.

Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like activity of the proteasome.

Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits beta5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138+ tumor cells. Although specific inhibition of either LMP7 or beta5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both beta5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2alpha suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors.

Mural cell associated VEGF is required for organotypic vessel formation.

BACKGROUND: Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics. METHODS AND FINDINGS: To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF. CONCLUSIONS: These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.

Enhanced gene expression from retroviral vectors.

BACKGROUND: Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter. RESULTS: By exploiting a new method of producing the retroviral genome in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods. We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow in vitro transcription of the retroviral genome by T7 RNA polymerase. When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression - up to 3.5 times greater than conventional retroviral LTR-driven expression. CONCLUSION: Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with in vitro transcribed retroviral genomic RNA. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. Furthermore, because the in vitro transcripts are not translated within the packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.

Expanding the spectrum of genetic elements transferable by retroviral vectors.

Retroviral vectors are powerful tools to study gene function. However, conventional methods require a cellular transcription step to generate the genomic RNA for viral production. This limits the scope of genetic elements that may be transferred by these vectors, excluding many key gene regulatory signals, including RNA editing motifs, alternative splicing, and various promoter/enhancer constellations, as well as cytotoxic genes. To address this problem, we devised a simple approach where in vitro-synthesized vector genomic RNA is transfected into the cytoplasm of a packaging cell, allowing immediate viral particle assembly. We demonstrate that high-titer retroviruses that efficiently transduce mammalian cell lines and primary cells are readily generated. Importantly, we show that an intron-containing expression cassette can be transferred by this method, leading to increased expression levels in the target cell. Further, we demonstrate that the cap structure is not required for retroviral packaging, thus avoiding translation of vector-encoded genes in the packaging cell. This allows the retroviral transfer of cytotoxic genes or proteins that otherwise inhibit viral production.

In vivo optical imaging of acute myeloid leukemia by green fluorescent protein: time-domain autofluorescence decoupling, fluorophore quantification, and localization.

Human xenografts of acute myeloid leukemia (AML) in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice result in disease states of diffuse, nonpalpable tissue infiltrates exhibiting a variable disease course, with some animals not developing a disease phenotype. Thus, disease staging and, more critically, quantification of preclinical therapeutic effect in these models are particularly difficult. In this study, we present the generation of a green fluorescent protein (GFP)-labeled human leukemic cell line, NB4, and validate the potential of a time-domain imager fitted with a 470 nm picosecond pulsed laser diode to decouple GFP fluorescence from autofluorescence on the basis of fluorescence lifetime and thus determine the depth and relative concentration of GFP inclusions in phantoms of homogeneous and heterogeneous optical properties. Subsequently, we developed an optical imageable human xenograft model of NB4-GFP AML and illustrate early disease detection, depth discrimination of leukemic infiltrates, and longitudinal monitoring of disease course employing time-domain optical imaging. We conclude that early disease detection through use of time-domain imaging in this initially slowly progressing AML xenograft model permits accurate disease staging and should aid in future preclinical development of therapeutics for AML.

MicroRNAs in tumorigenesis.

Emerging evidence suggests a class of non-coding RNAs termed microRNAs (miRNAs) play a key role in cancer. Since their original discovery in C. elegans in 1993 it has become evident that miRNAs are responsible for an entirely new mechanism of post-transcriptional gene regulation. miRNA expression is widespread in mammalian cells and notably altered in several cancer types. miRNA expression patterns correlate with several aspects of tumorigenesis and miRNA loci have been mapped to frequently altered cancer-associated genomic regions. Inhibition or augmentation of miRNA expression in cancer cells impacts gene expression and affects cell proliferation and survival. Hence, cancer-associated miRNAs may be regarded as a new class of non-coding tumour suppressors and oncogenes capable of regulating several key signalling pathways.

RNAi screening for therapeutic targets in human malignancies.

The advent of RNA interference (RNAi) based library screening approaches has sparked a surge in loss-of-function genetic screens. Several recent screens have aimed to identify novel regulators of cancer-related phenotypes. These employ various tumor cell types to model malignant cell functions and use different RNAi effector library approaches to reveal a cache of novel tumor regulators. This review surveys recent RNAi screens conducted in transformed human cells.

Drug target discovery using retroviruses.

Contemporary drug target discovery relies on a continuum of genetic and chemical-based screening technologies. These approaches conflate pharmaceutical and genetic principles, providing a conceptual platform that links dominant genetics with drug action. Thus, phenotypic genetic screens using vector-expressed dominant genetic effectors - trans-acting molecules that modulate gene function, such as peptides or RNA interference triggers - can reveal genes whose inhibition engenders a therapeutic effect. The correlation of this genetic inhibition with a specific protein activity defines a drug target candidate. Retroviruses provide a unique opportunity to stably deliver a variety of dominant genetic effectors to mammalian cells in a flexible predetermined fashion and are a favoured system for phenotypic screening. Here, the authors review recent innovations and approaches to therapeutic target discovery using retroviral vectors.