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Cancer-associated fibroblasts (CAF) are a prominent cell type within the tumor microenvironment (TME) where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease fibroblast activation protein (FAP) is expressed on the surface of highly protumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating protumorigenic CAFs will disrupt the cross-talk between components of TME resulting in cancer cell death and immune infiltration. This hypothesis, however, has never been directly proven. To eliminate FAP-expressing CAFs, we developed an antibody-drug conjugate using our anti-FAP antibody, huB12, coupled to a monomethyl auristatin E (huB12-MMAE) payload. After determining that huB12 was an effective targeting vector, we found that huB12-MMAE potently eliminated FAP-expressing cells as monocultures in vitro and significantly prolonged survival in vivo using a xenograft engineered to overexpress FAP. We investigated the effects of selectively eliminating CAFs using a layered, open microfluidic cell coculture platform, known as the Stacks. Analysis of mRNA and protein expression found that treatment with huB12-MMAE resulted in the increased secretion of the proinflammatory cytokines IL6 and IL8 by CAFs and an associated increase in expression of proinflammatory genes in cancer cells. We also detected increased secretion of CSF1, a cytokine involved in myeloid recruitment and differentiation. Our findings suggest that the mechanism of FAP-targeted therapies is through effects on the immune microenvironment and antitumor immune response. SIGNIFICANCE: The direct elimination of FAP-expressing CAFs disrupts the cross-talk with cancer cells leading to a proinflammatory response and alterations in the immune microenvironment and antitumor immune response.
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Fibroblastos Asociados al Cáncer , Endopeptidasas , Inmunoconjugados , Microambiente Tumoral , Humanos , Animales , Inmunoconjugados/farmacología , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/patología , Fibroblastos Asociados al Cáncer/inmunología , Ratones , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Endopeptidasas/genética , Endopeptidasas/metabolismo , Línea Celular Tumoral , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Gelatinasas/metabolismo , Gelatinasas/genética , Oligopéptidos/farmacología , FemeninoRESUMEN
Histopathologic diagnosis and classification of cancer plays a critical role in guiding treatment. Advances in next-generation sequencing have ushered in new complementary molecular frameworks. However, existing approaches do not independently assess both site-of-origin (e.g. prostate) and lineage (e.g. adenocarcinoma) and have minimal validation in metastatic disease, where classification is more difficult. Utilizing gradient-boosted machine learning, we developed ATLAS, a pair of separate AI Tumor Lineage and Site-of-origin models from RNA expression data on 8249 tumor samples. We assessed performance independently in 10,376 total tumor samples, including 1490 metastatic samples, achieving an accuracy of 91.4% for cancer site-of-origin and 97.1% for cancer lineage. High confidence predictions (encompassing the majority of cases) were accurate 98-99% of the time in both localized and remarkably even in metastatic samples. We also identified emergent properties of our lineage scores for tumor types on which the model was never trained (zero-shot learning). Adenocarcinoma/sarcoma lineage scores differentiated epithelioid from biphasic/sarcomatoid mesothelioma. Also, predicted lineage de-differentiation identified neuroendocrine/small cell tumors and was associated with poor outcomes across tumor types. Our platform-independent single-sample approach can be easily translated to existing RNA-seq platforms. ATLAS can complement and guide traditional histopathologic assessment in challenging situations and tumors of unknown primary.
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Adenocarcinoma , Mesotelioma Maligno , Tumores Neuroendocrinos , Masculino , Humanos , Aprendizaje Automático , Adenocarcinoma/diagnóstico , Adenocarcinoma/genéticaRESUMEN
Poly-ADP ribose polymerase inhibitors (PARPi) are an emerging therapeutic option for the treatment of prostate cancer. Their primary mechanism of action is via induction of synthetic lethality in cells with underlying deficiencies in homologous recombination repair (HRR). In men with metastatic castrate-resistant prostate cancer (mCRPC) and select HRR pathway alterations, PARPi treatment has been shown to induce objective tumor responses as well as improve progression free and overall survival. Presently, there are two PARPi, olaparib and rucaparib, that are FDA approved in the treatment of mCRPC. Ongoing research is focused on identifying which HRR alterations are best suited to predict response to PARPi so that these therapies can be most effectively utilized in the clinic. While resistance to PARPi remains a concern, combination therapies may represent a mechanism to overcome or delay resistance.
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BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype, the presence of which changes the prognosis and management of metastatic prostate cancer.MethodsWe performed analytical validation of a Circulating Tumor Cell (CTC) multiplex RNA qPCR assay to identify the limit of quantification (LOQ) in cell lines, synthetic cDNA, and patient samples. We next profiled 116 longitudinal samples from a prospectively collected institutional cohort of 17 patients with metastatic prostate cancer (7 NEPC, 10 adenocarcinoma) as well as 265 samples from 139 patients enrolled in 3 adenocarcinoma phase II trials of androgen receptor signaling inhibitors (ARSIs). We assessed a NEPC liquid biomarker via the presence of neuroendocrine markers and the absence of androgen receptor (AR) target genes.ResultsUsing the analytical validation LOQ, liquid biomarker NEPC detection in the longitudinal cohort had a per-sample sensitivity of 51.35% and a specificity of 91.14%. However, when we incorporated the serial information from multiple liquid biopsies per patient, a unique aspect of this study, the per-patient predictions were 100% accurate, with a receiver-operating-curve (ROC) AUC of 1. In the adenocarcinoma ARSI trials, the presence of neuroendocrine markers, even while AR target gene expression was retained, was a strong negative prognostic factor.ConclusionOur analytically validated CTC biomarker can detect NEPC with high diagnostic accuracy when leveraging serial samples that are only feasible using liquid biopsies. Patients with expression of NE genes while retaining AR-target gene expression may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype.FundingNIH (DP2 OD030734, 1UH2CA260389, R01CA247479, and P30 CA014520), Department of Defense (PC190039 and PC200334), and Prostate Cancer Foundation (Movember Foundation - PCF Challenge Award).
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Adenocarcinoma , Neoplasias de la Próstata , Humanos , Masculino , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/patología , Biomarcadores , Transducción de Señal , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
Neovascularization, the formation of new blood vessels, has received much research attention due to its implications for physiological processes and diseases. Most studies using traditional in vitro and in vivo platforms find challenges in recapitulating key cellular and mechanical cues of the neovascularization processes. Microfluidic in vitro models have been presented as an alternative to these limitations due to their capacity to leverage microscale physics to control cell organization and integrate biochemical and mechanical cues, such as shear stress, cell-cell interactions, or nutrient gradients, making them an ideal option for recapitulating organ physiology. Much has been written about the use of microfluidics in vascular biology models from an engineering perspective. However, a review introducing the different models, components and progress for new potential adopters of these technologies was absent in the literature. Therefore, this paper aims to approach the use of microfluidic technologies in vascular biology from a perspective of biological hallmarks to be studied and written for a wide audience ranging from clinicians to engineers. Here we review applications of microfluidics in vascular biology research, starting with design considerations and fabrication techniques. After that, we review the state of the art in recapitulating angiogenesis and vasculogenesis, according to the hallmarks recapitulated and complexity of the models. Finally, we discuss emerging research areas in neovascularization, such as drug discovery, and potential future directions.
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Microfluídica , Neovascularización Patológica , Biología , Comunicación Celular , Descubrimiento de Drogas , Humanos , Microfluídica/métodosRESUMEN
The tumor microenvironment (TME) is a complex network of non-malignant cells and stroma that perform a wide array of vital roles in tumor growth, immune evasion, metastasis, and therapeutic resistance. These highly diverse roles have been shown to be critically important to the progression of cancers and have already shown potential as therapeutic targets. Therefore, there has been a tremendous push to elucidate the pathways that underlie these roles and to develop new TME-directed therapies for cancer treatment. Unfortunately, TME-focused research has been limited by a lack of translational in vitro culture platforms that can model this highly complex niche and can support the integrated analysis of cell biology and function. In the current study, we investigate whether an independently developed reconfigurable microfluidic platform, known as Stacks, can address the critical need for translational multi-cellular tumor models and integrated analytics in TME research. We present data on multi-cellular culture of primary human cells in Stacks as well as the orthogonal analysis of cellular polarization, differentiation, migration, and cytotoxicity in this reconfigurable system. These expanded capabilities of Stacks are highly relevant to the cancer research community with the potential to enhance clinical translation of pre-clinical TME studies and to yield novel biological insight into TME crosstalk, metastasis, and responses to novel drug combinations or immune therapies.
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Neoplasias , Microambiente Tumoral , Técnicas de Cultivo de Célula , Humanos , Microfluídica , Neoplasias/patologíaRESUMEN
PURPOSE: Liquid biopsies in metastatic renal cell carcinoma (mRCC) provide a unique approach to understand the molecular basis of treatment response and resistance. This is particularly important in the context of immunotherapies, which target key immune-tumor interactions. Unlike metastatic tissue biopsies, serial real-time profiling of mRCC is feasible with our noninvasive circulating tumor cell (CTC) approach. METHODS: We collected 457 longitudinal liquid biopsies from 104 patients with mRCC enrolled in one of two studies, either a prospective cohort or a phase II multicenter adaptive immunotherapy trial. Using a novel CTC capture and automated microscopy platform, we profiled CTC enumeration and expression of HLA I and programmed cell death-ligand 1 (PD-L1). Given their diametric immunological roles, we focused on the HLA I to PD-L1 ratio (HP ratio). RESULTS: Patients with radiographic responses showed significantly lower CTC abundances throughout treatment. Furthermore, patients whose CTC enumeration trajectory was in the highest quartile (> 0.12 CTCs/mL annually) had shorter overall survival (median 17.0 months v 21.1 months, P < .001). Throughout treatment, the HP ratio decreased in patients receiving immunotherapy but not in patients receiving tyrosine kinase inhibitors. Patients with an HP ratio trajectory in the highest quartile (≥ 0.0012 annually) displayed significantly shorter overall survival (median 18.4 months v 21.2 months, P = .003). CONCLUSION: In the first large longitudinal CTC study in mRCC to date to our knowledge, we identified the prognostic importance of CTC enumeration and the change over time of both CTC enumeration and the HP ratio. These insights into changes in both tumor burden and the molecular profile of tumor cells in response to different treatments provide potential biomarkers to predict and monitor response to immunotherapy in mRCC.
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Carcinoma de Células Renales , Neoplasias Renales , Células Neoplásicas Circulantes , Humanos , Células Neoplásicas Circulantes/patología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/terapia , Antígeno B7-H1/metabolismo , Estudios Prospectivos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/terapia , PronósticoRESUMEN
Bone metastases represent a lethal condition that frequently occurs in solid tumors such as prostate, breast, lung, and renal cell carcinomas, and increase the risk of skeletal-related events (SREs) including pain, pathologic fractures, and spinal cord compression. This unique metastatic niche consists of a multicellular complex that cancer cells co-opt to engender bone remodeling, immune suppression, and stromal-mediated therapeutic resistance. This review comprehensively discusses clinical challenges of bone metastases, novel preclinical models of the bone and bone marrow microenviroment, and crucial signaling pathways active in bone homeostasis and metastatic niche. These studies establish the context to summarize the current state of investigational agents targeting BM, and approaches to improve BM-targeting therapies. Finally, we discuss opportunities to advance research in bone and bone marrow microenvironments by increasing complexity of humanized preclinical models and fostering interdisciplinary collaborations to translational research in this challenging metastatic niche.
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Prostate Cancer (PC) is a disease with remarkable tumor heterogeneity that often manifests in significant intra-patient variability with regards to clinical outcomes and treatment response. Commonly available PC cell lines do not accurately reflect the complexity of this disease and there is critical need for development of new models to recapitulate the intricate hierarchy of tumor pathogenesis. In current study, we established ex vivo primary patient-derived cancer organoid (PDCO) cultures from prostatectomy specimens of patients with locally advanced PC. We then performed a comprehensive multi-parameter characterization of the cellular composition utilizing a novel approach for live-cell staining and direct imaging in the integrated microfluidic Stacks device. Using orthogonal flow cytometry analysis, we demonstrate that primary PDCOs maintain distinct subsets of epithelial cells throughout culture and that these cells conserve expression of androgen receptor (AR)-related elements. Furthermore, to confirm the tumor-origin of the PDCOs we have analyzed the expression of PC-associated epigenetic biomarkers including promoter methylation of the GSTP1, RASSF1 and APC and RARb genes by employing a novel microfluidic rare-event screening protocol. These results demonstrate that this ex vivo PDCO model recapitulates the complexity of the epithelial tumor microenvironment of multifocal PC using orthogonal analyses. Furthermore, we propose to leverage the Stacks microfluidic device as a high-throughput, translational platform to interrogate phenotypic and molecular endpoints with the capacity to incorporate a complex tumor microenvironment.
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Organoides/fisiología , Neoplasias de la Próstata/patología , Receptores Androgénicos/fisiología , Línea Celular Tumoral , Humanos , Receptores de Hialuranos/análisis , Dispositivos Laboratorio en un Chip , Masculino , Organoides/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Transducción de Señal/fisiología , Microambiente TumoralRESUMEN
Bone marrow (BM) is a primary metastatic site in prostate cancer (PC) and bone invasion is considered incurable. T cell-mediated immune surveillance is essential in controlling both tumorigenesis and initiation of metastases. Beside tropism, dissemination of PC cells to the BM may be facilitated by defects in BM immune homeostasis predisposing this niche to colonization. To evaluate the BM immune microenvironment in locally advanced, non-metastatic PC, we performed flow cytometry analysis of myeloid and lymphoid subsets in BM aspirates and peripheral blood collected during prostatectomy. Healthy BM aspirates served to establish a reference range for comparison. We found alterations in BM immune composition of PC patients, including an increased CD4/CD8 ratio, enrichment of CD4+ T cells, increased CD56+CD3+ NKT and CD56+CD3- NK yields compared to healthy controls. The lymphoid phenotype remained comparable regarding T cell activation and chemokine receptor-based polarization patterns. Additionally, we found increased B7H3 expression in the myeloid monocyte/macrophage subset and decreased DC infiltration in BM of PC patients. These findings suggest that alterations in the immune milieu may limit immune surveillance that compromise the ability of the BM microenvironment to prevent tumor dissemination, and predispose development of bone metastases in a subset of patients with localized PC.
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Tumor-associated macrophages (TAMs) regulate an array of tumor functions and have critical roles in both the progression and the eradication of cancer. Numerous therapies targeting TAMs are under development in cancer and many have demonstrated success at the preclinical and clinical levels. Most of these therapies fall within 3 main categories: systemic depletion of TAMs, inhibition of TAM recruitment and polarization, and promoting the antitumor functions of TAMs. In this article, the rationale behind these various therapies and approaches is reviewed along with supporting preclinical and clinical data.
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Macrófagos/efectos de los fármacos , Neoplasias/terapia , Humanos , Microambiente TumoralRESUMEN
Micromilling is an underutilized technique for fabricating microfluidic platforms that is well-suited for the diverse needs of the biologic community. This technique, however, produces culture surfaces that are considerably rougher than in commercially available culture platforms and the hydrophilicity of these surfaces can vary considerably depending on the choice of material. In this study, we evaluated the impact of surface topography and hydrophilicity in milled microfluidic devices on the cellular phenotype and function of primary human macrophages. We found that the rough culture surface within micromilled systems affected the phenotype of macrophages cultured in these devices. However, the presence, type, and magnitude of this effect was dependent on the surface hydrophilicity as well as exposure to chemical polarization signals. These findings confirm that while milled microfluidic systems are an effective platform for culture and analysis of primary macrophages, the topography and hydrophilicity of the culture surface within these systems should be considered in the planning and analysis of any macrophage experiments in which phenotype is relevant.
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Interacciones Hidrofóbicas e Hidrofílicas , Dispositivos Laboratorio en un Chip , Macrófagos/citología , Diferenciación Celular , Polaridad Celular , Proliferación Celular , Regulación de la Expresión Génica , Humanos , Macrófagos/metabolismo , Fenómenos Mecánicos , Propiedades de SuperficieRESUMEN
Immune tolerance to self-antigens can limit robust anti-tumor immune responses in the use of tumor vaccines. Expression of novel tumor associated antigens can improve immune recognition and lysis of tumor cells. The cancer-testis antigen (CTA) family of proteins has been hypothesized to be an ideal class of antigens due to tumor-restricted expression, a subset of which have been found to induce antibody responses in patients with prostate disease. We demonstrate that CTA expression is highly inducible in five different Prostate Cancer (PC) cell lines using a hypomethylating agent 5-Aza-2'-deoxycytidine (5AZA) and/or a histone deacetylase inhibitor LBH589. These CTAs include NY-ESO1, multiple members of the MAGE and SSX families and NY-SAR35. A subset of CTAs is synergistically induced by the combination of 5AZA and LBH589. We developed an ex vivo organ culture using human PC biopsies for ex vivo drug treatments to evaluate these agents in clinical samples. These assays found significant induction of SSX2 in 9/9 distinct patient samples and NY-SAR35 in 7/9 samples. Further, we identify expression of SSX2 in circulating tumor cells (CTC) from patients with advanced PC. These results indicate that epigenetic modifying agents can induce expression of a broad range of neoantigens in human PC and may serve as a useful adjunctive therapy with novel tumor vaccines and checkpoint inhibitors.
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Antígenos de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias de la Próstata/genética , Testículo/metabolismo , Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Decitabina , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Masculino , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Células Neoplásicas Circulantes/metabolismo , Panobinostat , Neoplasias de la Próstata/patología , Proteínas Represoras/genéticaRESUMEN
The ability to evade host immune surveillance is critical for the survival of tumor cells and is correlated with poor clinical outcomes. Many tumor types have been found to downregulate expression of genes involved in antigen production, processing and presentation to evade immune detection. Recent findings suggest that the mechanisms underlying these immune evasion phenomena extend beyond alterations in DNA sequence to include epigenetic modifications of DNA and associated proteins, including hypermethylation of DNA and altered histone acetylation patterns. This review will summarize alterations in antigen presentation machinery identified in malignant cells, epigenetic mechanisms that can be employed in the downregulation of genes relevant for antigen presentation and translational strategies to target these processes to enhance the efficacy of antitumor immunotherapies.