Stromal Interactions Research Articles

Radiosynthesis and Preclinical Evaluation of Bispecific PSMA/FAP Heterodimers for Tumor Imaging.

Due to tumor heterogeneity and complex tumor–stromal interactions in multicellular systems, the efficiency of monospecific tracers for tumor diagnosis and therapy is currently limited. In light of the evidence of prostate-specific membrane antigen (PSMA) overexpression in tumor cells and fibroblast activation protein (FAP) upregulation in the tumor stroma, heterodimer dual targeting PSMA and FAP may have the potential to improve tumor diagnosis. Herein, we described the radiosynthesis, in vitro characterization, and micro-PET/CT imaging of two novel 18F-labeled bispecific PSMA/FAP heterodimers. 18F-labeled heterodimers showed high specificity and affinity targeting to PSMA and FAP in vitro and in vivo. Compared with the monospecific tracers [18F]AlF-PSMA-BCH and [18F]FAPI-42, both 18F-labeled heterodimers exhibited better tumor uptake in tumor-bearing mice. Their favorable characterizations such as convenient synthesis, high tumor uptake, and favorable pharmacokinetic profile could lead to their future applications as bispecific radiotracers for clinical cancer imaging.

Distinct Response of Circulating microRNAs to the Treatment of Pancreatic Cancer Xenografts with FGFR and ALK Kinase Inhibitors.

Simple SummaryPancreatic cancer is among the top three leading causes of cancer-related death in the United States, mainly due to late diagnosis after the disease has already spread to other organs. Hence, timely and efficient treatments are necessary to improve outcomes. In this study, we explored the use of circulating microRNAs (miRs) collected from the peripheral blood of pancreatic cancer-bearing mice for assessment of treatment efficacy early in the treatment schedule. MicroRNA changes in serum samples precede histologic changes during the treatments, suggesting the use of circulating microRNA as early indicators of response to treatment. These easily-accessible biomarkers can be sampled repeatedly and could guide timely treatment decisions.Pancreatic adenocarcinoma is typically detected at a late stage and thus shows only limited sensitivity to treatment, making it one of the deadliest malignancies. In this study, we evaluate changes in microRNA (miR) patterns in peripheral blood as a potential readout of treatment responses of pancreatic cancer to inhibitors that target tumor–stroma interactions. Mice with pancreatic cancer cell (COLO357PL) xenografts were treated with inhibitors of either fibroblast growth factor receptor kinase (FGFR; PD173074) or anaplastic lymphoma kinase receptor (ALK; TAE684). While both treatments inhibited tumor angiogenesis, signal transduction, and mitogenesis to a similar extent, they resulted in distinct changes in circulating miR signatures. Comparison of the miR pattern in the tumor versus that in circulation showed that the inhibitors can be distinguished by their differential impact on tumor-derived miRs as well as host-derived circulating miRs. Distinct signatures that include circulating miR-1 and miR-22 are associated with the efficacy of ALK and FGFR inhibition, respectively. We propose that monitoring changes in circulating miR profiles can provide an early signature of treatment response or resistance to pathway-targeted drugs, and thus provide a non-invasive measurement to rapidly assess the efficacy of candidate therapies.

Inside the Joint of Inflammatory Arthritis Patients: Handling and Processing of Synovial Tissue Biopsies for High Throughput Analysis.

Inflammatory arthritis is a chronic systemic autoimmune disease of unknown etiology, which affects the joints. If untreated, these diseases can have a detrimental effect on the patient's quality of life, leading to disabilities, and therefore, exhibit a significant socioeconomic impact and burden. While studies of immune cell populations in arthritis patient's peripheral blood have been informative regarding potential immune cell dysfunction and possible patient stratification, there are considerable limitations in identifying the early events that lead to synovial inflammation. The joint, as the site of inflammation and the local microenvironment, exhibit unique characteristics that contribute to disease pathogenesis. Understanding the contribution of immune and stromal cell interactions within the inflamed joint has been met with several technical challenges. Additionally, the limited availability of synovial tissue biopsies is a key incentive for the utilization of high-throughput techniques in order to maximize information gain. This review aims to provide an overview of key methods and novel techniques that are used in the handling, processing and analysis of synovial tissue biopsies and the potential synergy between these techniques. Herein, we describe the utilization of high dimensionality flow cytometric analysis, single cell RNA sequencing, ex vivo functional assays and non-intrusive metabolic characterization of synovial cells on a single cell level based on fluorescent lifetime imaging microscopy. Additionally, we recommend important points of consideration regarding the effect of different storage and handling techniques on downstream analysis of synovial tissue samples. The introduction of new powerful techniques in the study of synovial tissue inflammation, brings new challenges but importantly, significant opportunities. Implementation of novel approaches will accelerate our path toward understanding of the mechanisms involved in the pathogenesis of inflammatory arthritis and lead to the identification of new avenues of therapeutic intervention.

Megakaryocyte–stromal cell interactions: Effect on megakaryocyte proliferation, proplatelet production, and survival

Bone marrow stromal cells provide a proper environment for the development of hematologic lineages. The incorporation of different stromal cells into in vitro culture systems would be an attractive model to study megakaryopoiesis and thrombopoiesis. Our objective was to evaluate the participation of different types of stromal cells in in vitro megakaryopoiesis, thrombopoiesis, and megakaryocyte (MK) survival. CD34-positive progenitors from umbilical cord blood were differentiated into MK precursors and then cocultured with umbilical vein endothelial cells (HUVECs), bone marrow mesenchymal stem cells (MSCs), skin fibroblasts (SFs) (all human), or the mouse fibroblast cell line L929. The number of MKs (CD61-positive cells) was increased in the presence of HUVECs and SFs, whereas L929 cells decreased total and mature MK counts. With respect to thrombopoiesis, HUVECs increased proplatelet (PP)-producing MKs, while MSCs, L929 cells, and SFs had the opposite effect (immunofluorescence staining and microscopic analysis). MK survival was enhanced in MSC and SF co-cultures, as assessed by evaluation of pyknotic nuclei. However, HUVECs and L929 did not prevent apoptosis of MKs. Reciprocally, the cloning efficiency of MSCs was decreased in the presence of MKs, while the ability of stromal cells (either MSCs or SFs) to produce the extracellular matrix proteins type III collagen, fibronectin, dermatan sulfate, heparan sulfate, and prolyl 4-hydroxylase subunit β was preserved. These data indicate that each stromal cell type performs distinctive functions that differentially modulate MK growth and platelet production and, at the same time, that MKs also modify stromal cell behavior. Overall, our results highlight the relevance of considering the influence of stromal cells in MK research as well as the close interplay of different cell types within the bone marrow milieu.

Alcohol and Head and Neck Cancer: Updates on the Role of Oxidative Stress, Genetic, Epigenetics, Oral Microbiota, Antioxidants, and Alkylating Agents.

Head and neck cancer (HNC) concerns more than 890,000 patients worldwide annually and is associated with the advanced stage at presentation and heavy outcomes. Alcohol drinking, together with tobacco smoking, and human papillomavirus infection are the main recognized risk factors. The tumorigenesis of HNC represents an intricate sequential process that implicates a gradual acquisition of genetic and epigenetics alterations targeting crucial pathways regulating cell growth, motility, and stromal interactions. Tumor microenvironment and growth factors also play a major role in HNC. Alcohol toxicity is caused both directly by ethanol and indirectly by its metabolic products, with the involvement of the oral microbiota and oxidative stress; alcohol might enhance the exposure of epithelial cells to carcinogens, causing epigenetic modifications, DNA damage, and inaccurate DNA repair with the formation of DNA adducts. Long-term markers of alcohol consumption, especially those detected in the hair, may provide crucial information on the real alcohol drinking of HNC patients. Strategies for prevention could include food supplements as polyphenols, and alkylating drugs as therapy that play a key role in HNC management. Indeed, polyphenols throughout their antioxidant and anti-inflammatory actions may counteract or limit the toxic effect of alcohol whereas alkylating agents inhibiting cancer cells’ growth could reduce the carcinogenic damage induced by alcohol. Despite the established association between alcohol and HNC, a concerning pattern of alcohol consumption in survivors of HNC has been shown. It is of primary importance to increase the awareness of cancer risks associated with alcohol consumption, both in oncologic patients and the general population, to provide advice for reducing HNC prevalence and complications.

Increased Expression and Altered Cellular Localization of Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) Are Associated with Prostate Cancer Progression.

Simple SummaryProstate cancer (PCa) is one of the most frequently diagnosed malignancies in men. PCa is primarily regulated by androgens, but other mechanisms, such as fibroblast growth factor receptor (FGFR) signaling, are also involved. In some patients, PCa relapses after surgical removal of prostate, and androgen deprivation therapy (ADT) is used as the first-line treatment. Unfortunately, the patients often lose response to ADT and progress by other mechanisms to castration-resistant, currently non-curable PCa. In our study, we aimed to identify better diagnostic markers and therapeutic targets against PCa. We analyzed patient PCa tissue samples from radical prostatectomies and biopsies, and used physiologically relevant 3D organoids and mouse xenografts to study FGFR signaling in PCa. We found that FGFRL1, a protein belonging to the FGFR family, plays a role in PCa. Our results suggest that FGFRL1 has significant effects on PCa progression and has potential as a prognostic biomarker.Fibroblast growth factor receptors (FGFRs) 1–4 are involved in prostate cancer (PCa) regulation, but the role of FGFR-like 1 (FGFRL1) in PCa is unclear. FGFRL1 expression was studied by qRT-PCR and immunohistochemistry of patient tissue microarrays (TMAs) and correlated with clinical patient data. The effects of FGFRL1 knockdown (KD) in PC3M were studied in in vitro culture models and in mouse xenograft tumors. Our results showed that FGFRL1 was significantly upregulated in PCa. The level of membranous FGFRL1 was negatively associated with high Gleason scores (GSs) and Ki67, while increased cytoplasmic and nuclear FGFRL1 showed a positive correlation. Cox regression analysis indicated that nuclear FGFRL1 was an independent prognostic marker for biochemical recurrence after radical prostatectomy. Functional studies indicated that FGFRL1-KD in PC3M cells increases FGFR signaling, whereas FGFRL1 overexpression attenuates it, supporting decoy receptor actions of membrane-localized FGFRL1. In accordance with clinical data, FGFRL1-KD markedly suppressed PC3M xenograft growth. Transcriptomics of FGFRL1-KD cells and xenografts revealed major changes in genes regulating differentiation, ECM turnover, and tumor–stromal interactions associated with decreased growth in FGFRL1-KD xenografts. Our results suggest that FGFRL1 upregulation and altered cellular compartmentalization contribute to PCa progression. The nuclear FGFRL1 could serve as a prognostic marker for PCa patients.

Crosstalk between cancer-associated fibroblasts and immune cells in peritoneal metastasis: inhibition in the migration of M2 macrophages and mast cells by Tranilast

BackgroundThe role of tumor–stroma interactions in tumor immune microenvironment (TME) is attracting attention. We have previously reported that cancer-associated fibroblasts (CAFs) contribute to the progression of peritoneal metastasis (PM) in gastric cancer (GC), and M2 macrophages and mast cells also contribute to TME of PM. To elucidate the role of CAFs in TME, we established an immunocompetent mouse PM model with fibrosis, which reflects clinical features of TME. However, the involvement of CAFs in the immunosuppressive microenvironment remains unclear. In this study, we investigated the efficacy of Tranilast at modifying this immune tolerance by suppressing CAFs.MethodsThe interaction between mouse myofibroblast cell line LmcMF and mouse GC cell line YTN16 on M2 macrophage migration was investigated, and the inhibitory effect of Tranilast was examined in vitro. Using C57BL/6J mouse PM model established using YTN16 with co-inoculation of LmcMF, TME of resected PM treated with or without Tranilast was analyzed by immunohistochemistry.ResultsThe addition of YTN16 cell-conditioned medium to LmcMF cells enhanced CXCL12 expression and stimulated M2 macrophage migration, whereas Tranilast inhibited the migration ability of M2 macrophages by suppressing CXCL12 secretion from LmcMF. In PM model, Tranilast inhibited tumor growth and fibrosis, M2 macrophage, and mast cell infiltration and significantly promoted CD8 + lymphocyte infiltration into the tumor, leading to apoptosis of cancer cells by an immune response.ConclusionTranilast improved the immunosuppressive microenvironment by inhibiting CAF function in a mouse PM model. Tranilast is thus a promising candidate for the treatment of PM.

Recapitulating the Angiogenic Switch in a Hydrogel-Based 3D In Vitro Tumor-Stroma Model.

To ensure nutrient and oxygen supply, tumors beyond a size of 1–2 mm3 need a connection to the vascular system. Thus, tumor cells modify physiological tissue homeostasis by secreting inflammatory and angiogenic cytokines. This leads to the activation of the tumor microenvironment and the turning of the angiogenic switch, resulting in tumor vascularization and growth. To inhibit tumor growth by developing efficient anti-angiogenic therapies, an in depth understanding of the molecular mechanism initiating angiogenesis is essential. Yet so far, predominantly 2D cell cultures or animal models have been used to clarify the interactions within the tumor stroma, resulting in poor transferability of the data obtained to the in vivo situation. Consequently, there is an abundant need for complex, humanized, 3D models in vitro. We established a dextran-hydrogel-based 3D organotypic in vitro model containing microtumor spheroids, macrophages, neutrophils, fibroblasts and endothelial cells, allowing for the analysis of tumor–stroma interactions in a controlled and modifiable environment. During the cultivation period of 21 days, the microtumor spheroids in the model grew in size and endothelial cells formed elongated tubular structures resembling capillary vessels, that appeared to extend towards the tumor spheroids. The tubular structures exhibited complex bifurcations and expanded without adding external angiogenic factors such as VEGF to the culture. To allow high-throughput screening of therapeutic candidates, the 3D cell culture model was successfully miniaturized to a 96-well format, while still maintaining the same level of tumor spheroid growth and vascular sprouting. The quantification of VEGF in the conditioned medium of these cultures showed a continuous increase during the cultivation period, suggesting the contribution of endogenous VEGF to the induction of the angiogenic switch and vascular sprouting. Thus, this model is highly suitable as a testing platform for novel anticancer therapeutics targeting the tumor as well as the vascular compartment.

Hepatocellular Carcinoma Cells Are Protected From Immunolysis by Mesenchymal Stromal Cells Through Indoleamine 2,3 Dioxygenase.

B7 family proteins serve as checkpoint molecules that protect tumors from T cell mediated lysis. Tryptophan degrading enzymes indoleamine 2,3 dioxygenase (IDO) and tryptophan 2,3 dioxygenase (TDO) also induce T cell immune tolerance. However, little is known about the relative contribution of B7 molecules, tryptophan degrading enzymes, as well as the impact of tumor and stromal cell interactions to the development of immunosuppressive tumor microenvironment. To investigate such interactions, we used a tripartite model of human hepatocellular carcinoma cell line (HepG2) and mesenchymal stromal cells (MSCs) co-cultured with peripheral blood mononuclear cells (PBMCs). Co-culture of HepG2 cells and activated PBMCs demonstrate that HepG2 cells undergo PBMC mediated cytolysis, despite constitutive expression of B7-H3 and upregulation of PD-L1 by IFNγ. Knockdown of B7-H3, PD-L1 or IDO does not modulate PBMC mediated lysis of HepG2 cells. However, TNFα preactivation enhances lysis of HepG2 cells, and blocking of TNFα production from PBMCs protects HepG2 cells. On the other hand, MSCs protect HepG2 cells from PBMC mediated lysis, even in the presence of TNFα. Further investigation showed that MSC mediated protection is associated with the unique secretome profile of upregulated and downregulated cytokines and chemokines. IFNγ activated MSCs are superior to TNFα activated or control MSCs in protecting HepG2 cells. Blockade of IFNγ driven IDO activity completely abolishes the ability of MSCs to protect HepG2 cells from cytolysis by PBMCs. These results suggest that inhibition of IFNγ activation of IDO induction in stromal cells, combined with usage of TNFα, could be a novel immunotherapeutic strategy to induce regression of hepatocellular carcinoma.

Chromatin Remodeling and Associated Changes in Gene Expression Induced By Bone Marrow Stromal Cells Identify Features of High-Risk Multiple Myeloma

Introduction: Stromal cells in the bone marrow microenvironment maintain a complex bidirectional relationship with the malignant plasma cells and have been implicated in the growth and survival of multiple myeloma (MM) cells along with the development of drug resistance and disease progression. We hypothesized that the perpetual induction of gene expression changes similar to those induced by stromal interactions may lead to an aggressive disease phenotype and possible stromal independence. We investigated the chromatin remodeling and associated gene expression changes in malignant plasma cells induced by stromal cells and applied the findings to large sets of patient data.Methods: We interrogated the chromatin accessibility (ATAC-seq) and gene expression profiles (RNA-seq) of malignant plasma cells (INA6, MM1S, RPMI) after co-culture with stromal cells (HS5). We identified de novo accessible enhancers and associated them with their putative target genes within conserved topologically associating domains based on their proximity in the linear genome. The in vitro data were then applied to data from patients with newly diagnosed MM (n=559). Using proportional hazards regression and automated feature selection algorithms we selected 10 of the 68 overexpressed target genes based on their association with progression-free survival. We devised a dichotomous classifier based on the expression of these 10 genes and validated its independent prognostic significance and ability to predict therapeutic resistance in two additional patient cohorts (n=214 and n=635, respectively). We applied gene set enrichment analysis to interrogate whether the gene expression profile induced by these adverse stromal interactions recapitulates the transcriptional program of extramedullary malignant plasma cells using single-cell RNA-seq.Results: Co-culture of malignant plasma cells with stromal cells induced widespread chromatin remodeling including de novo accessibility of known cis-regulatory elements and the overexpression of their putative target genes (68 overexpressed genes with associated accessible enhancers, overlap p=1.05x10 -13). The expression of 10 of these 68 genes was independently associated with progression-free survival in the UAMS TT2/3 data set (n=559). A summary measure of the expression of these 10 genes was dichotomized to classify approximately 30% of patients as having adverse stromal interactions (ASI+). The 10-gene ASI+ classifier was associated with overall survival in three independent patient cohorts with newly diagnosed MM: UAMS TT2/3 (29% ASI+, HR 1.93, 95% CI 1.43-2.62, p<0.001, n=559), IFM 2009 (32% ASI+, HR 2.61, 95% CI 1.67-4.10, p<0.001, n=214), and MMRF IA16 (30% ASI+, HR 1.69, 95% CI 1.29-2.24, p<0.001, n=635). Its prognostic significance was independent of age, sex, ISS stage, elevated LDH, high-risk cytogenetics, and the high-risk transcriptomic classifiers (UAMS-70, EMC-92). The 10-gene ASI+ classifier predicted resistance to first-line therapy in general and to treatment with VRd in particular (OR 1.64, 95% CI 1.10-2.43, p=0.015, MMRF IA16, n=211). Gene set enrichment analysis demonstrated that the gene expression profile of patients with ASI+ recapitulates the transcriptional program of extramedullary malignant plasma cells (5376 circulating plasma cells, NES 1.41, FDR=0.027; 477 plasma cells from malignant effusions, NES 1.37, FDR=0.029). This extramedullary-like gene expression pattern translated into a higher prevalence of circulating plasma cells among newly diagnosed ASI+ patients (54% versus 35%, p=1.65x10 -5, MMRF IA16, n=629) and a higher incidence of bone disease and plasmacytoma progression during follow-up (HR 2.24, 95% CI 1.51-3.31, p<0.001, MMRF IA16, n=622).Conclusions: We identified permissive chromatin states and associated transcriptional programs induced by the interactions between MM cells and bone marrow stromal cells. The constitutive induction of genes in the malignant plasma cells of patients with newly diagnosed MM supported transcriptional programs associated with therapeutic resistance, accelerated disease dissemination, and impaired long-term survival. These changes may allow MM cells to acquire the capacity to sustain extramedullary growth. The development of novel therapeutic approaches to overcome these adverse stromal interactions remains an unmet need in patients with MM. DisclosuresMunshi: Adaptive Biotechnology: Consultancy; Karyopharm: Consultancy; Abbvie: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy.

Dynamic cell contacts between periportal mesenchyme and ductal epithelium act as a rheostat for liver cell proliferation.

SummaryIn the liver, ductal cells rarely proliferate during homeostasis but do so transiently after tissue injury. These cells can be expanded as organoids that recapitulate several of the cell-autonomous mechanisms of regeneration but lack the stromal interactions of the native tissue. Here, using organoid co-cultures that recapitulate the ductal-to-mesenchymal cell architecture of the portal tract, we demonstrate that a subpopulation of mouse periportal mesenchymal cells exerts dual control on proliferation of the epithelium. Ductal cell proliferation is either induced and sustained or, conversely, completely abolished, depending on the number of direct mesenchymal cell contacts, through a mechanism mediated, at least in part, by Notch signaling. Our findings expand the concept of the cellular niche in epithelial tissues, whereby not only soluble factors but also cell-cell contacts are the key regulatory cues involved in the control of cellular behaviors, suggesting a critical role for cell-cell contacts during regeneration.

Understanding Inflammation-associated Ophthalmic Pathologies: A Novel 3D Co-culture Model of Monocyte-myofibroblast Immunomodulation

ABSTRACT Purpose Inflammation is associated with, and may be causal of, a variety of ophthalmic pathologies. These pathologies are currently difficult to model in vitro because they involve complex interactions between the innate immune system, stromal cells, and other cells that normally maintain ocular tissue homeostasis. Using transscleral drainage channel fibrosis after glaucoma surgery as an example of inflammation-associated ocular fibrosis, we have assessed a simple but novel 3D cell culture system designed to reveal the immunomodulatory impacts of ocular connective tissue cells on monocytes, a major cellular component of the circulating immune system. Methods Primary human Tenon’s capsule fibroblasts derived from five unrelated patients were activated into myofibroblasts in 3D collagen matrices under isometric tension, with and without exposure to an inflammatory cytokine-enhanced milieu, and co-cultured with an immortalized human monocyte cell line (THP-1 cells). Quantitative PCR analyses were performed on 8 candidate genes to assess the impacts of inflammatory cytokines on the myofibroblasts and the monocytes in mono-cultures and compared to cells in co-culture to clearly distinguish any co-culture-induced impacts on gene expression. Results Our data indicate that both Tenon’s capsule myofibroblasts in 3D mono-culture and THP-1 monocytes in suspension mono-culture were responsive to inflammatory cytokine stimuli. Co-culture with Tenon’s capsule myofibroblasts significantly modulated the gene expression responses of THP-1 monocytes to inflammatory cytokine stimulation, indicative of an immunomodulatory “feedback” system between these cell types. Conclusion Our findings provide proof of principle for the use of simple 3D co-culture systems as a means to enhance our understanding of ocular stromal cell interactions with cells of the innate immune system and to provide more informative in vitro models of inflammation-associated ophthalmic pathologies.

Selected Articles from This Issue

Radium-223 dichloride is a radiopharmaceutical used to treat cancer that has metastasized to the bones, and whose effects are derived from both direct action of radiation on tumor cells as well as bystander effects caused by irradiation of both tumor and non-tumor cells in the bone microenvironment. Here, Canter and colleagues quantify the biological effects derived from both of these mechanisms of action. Direct effects from alpha particle irradiation (yellow arrows in the image at left) contribute to biological changes detected histologically in disseminated tumor cells and osteocytes within the range of the alpha particles. Bystander effects also contribute to biological changes in tumor cells beyond the range of the alpha particles emitted from the endosteal surface of the bone. These radiation-induced bystander effects can arise from tumor cells (white outlined arrow) and marrow cells (pink outlined arrow) within the range of alpha particles, and from bone cells (brown outlined arrow). The data suggest that bystander effects in the bone environment likely contribute to growth delay of disseminated tumor cells in bone marrow.Regulation of cancer stemness has emerged as a key tumor-intrinsic property underlying disease progression and therap. resistance. Recently, gain-of-function (GOF) mutations in p53 have been shown to promote increased stemness characteristics, but the mechanism for this is not well understood. Here, Ghatak and colleagues identify the microRNA miR-324-5p as the key effector of GOF mutant p53 that controls this phenomenon in non–small cell lung cancer (NSCLC) cells. GOF p53 isoforms expressed in NSCLC cells caused upregulation of c-Myc, which bound to and activated transcription of miR-324. The authors demonstrate that expression of CUEDC2—the direct target of miR-324-5p in this context—was ablated in cells expressing GOF p53 isoforms, and the loss of CUEDC2 expression was associated with activation of NF-κB and increased expression of stemness markers. CUEDC2 downregulation was further shown to be characteristic of human tumors bearing GOF p53 mutations. The authors conclude that TP53 mutations coupled with high miR-324-5p expression is associated with worse prognosis in lung cancer patients. Taken together, the data identify miR-324-5p as a novel epigenetic regulator of stemness properties in NSCLC.Balanced MDM2:p53 expression maintains proper cell cycle progression, whereas aberrant p53 activity is associated with developmental defects and pathologic states. Here, Mohibi and colleagues demonstrate that the RNA binding protein Zfp871 is a direct transcriptional target of p53 in vivo. Zfp871 functions to provoke a negative feedback loop regulating p53 activation by binding to the 3′UTR of MDM2 mRNA and stabilizing it. This interaction increases MDM2 expression and, by consequence, promotes degradation of p53. Mice deficient in Zfp871 were prone to early death, and heterozygous mice experienced a shortened lifespan and prominent steatohepatitis. The early death seen in Zfp871-KO mice, however, was ameliorated by ablation of Trp53. Gene expression analyses unveiled that p53 hyperactivation caused by Zfp871 loss was associated with alterations in lipid metabolism and inflammatory signaling as well as key developmental pathways. While the precise mechanism underlying the control of these pathways is yet to be determined, the data clearly demonstrate that precise control of p53 expression and activity—in part through the novel Zfp871-MDM2-p53 axis—is required to prevent the onset of pathologic conditions.Micro-metastatic lesions in the bone are often latent and can persist undetected for years in a quiescent state. However, the mechanisms by which rapidly proliferating tumor cells suddenly become quiescent upon metastasis are poorly defined. Here, Shupp and colleagues demonstrate that soluble signals from the bone metastatic microenvironment may play a key role in regulating this process. In particular, small extracellular vesicles (sEV) released by osteoblasts in proximity to breast cancer cells were shown to harbor factors such as miR-148a-3p, which had implications for breast cancer cell biology. Among other effects, the uptake of these sEV by metastatic breast cancer cells resulted in disruptions of both ERK1/2 signaling and cell cycle progression, thereby slowing cancer cell proliferation and contributing toward their latency. Taken together, the data elucidate a novel mechanism by which stromal cells influence cancer cell progression in the metastatic niche and underscore the importance of extracellular vesicles in mediating tumor–stromal cell interactions.

Epithelial–stromal cell interactions and extracellular matrix mechanics drive the formation of airway-mimetic tubular morphology in lung organoids

SummaryComplex human airway cellular organization where extracellular matrix (ECM) and epithelial and stromal lineages interact present challenges for organ study in vitro. Current in vitro lung models that focus on the lung epithelium do not represent complex airway morphology and cell-ECM interactions seen in vivo. Models including stromal populations often separate them via a semipermeable barrier precluding cell–cell interaction or the effect of ECM mechanics. We investigated the effect of stromal cells on basal epithelial cell-derived bronchosphere structure and function through a triple culture of human bronchial epithelial, lung fibroblast, and airway smooth muscle cells. Epithelial–stromal cross-talk resulted in epithelial cell-driven branching tubules with stromal cells surrounding epithelial cells termed bronchotubules. Agarose– Matrigel scaffold (Agrigel) formed a mechanically tuneable ECM, with adjustable viscoelasticity and stiffness enabling long-term tubule survival. Bronchotubule models may enable research into how epithelial–stromal cell and cell–ECM communication drive tissue patterning, repair, and development of disease.

Importance of lymphocyte-stromal cell interactions in autoimmune and inflammatory rheumatic diseases.

Interactions between lymphocytes and stromal cells have an important role in immune cell development and responses. During inflammation, stromal cells contribute to inflammation, from induction to chronicity or resolution, through direct cell interactions and through the secretion of pro-inflammatory and anti-inflammatory mediators. Stromal cells are imprinted with tissue-specific phenotypes and contribute to site-specific lymphocyte recruitment. During chronic inflammation, the modified pro-inflammatory microenvironment leads to changes in the stromal cells, which acquire a pathogenic phenotype. At the site of inflammation, infiltrating B cells and T cells interact with stromal cells. These interactions induce a plasma cell-like phenotype in B cells and T cells, associated with secretion of immunoglobulins and inflammatory cytokines, respectively. B cells and T cells also influence the stromal cells, inducing cell proliferation, molecular changes and cytokine production. This positive feedback loop contributes to disease chronicity. This Review describes the importance of these cell interactions in chronic inflammation, with a focus on human disease, using three selected autoimmune and inflammatory diseases: rheumatoid arthritis, psoriatic arthritis (and psoriasis) and systemic lupus erythematosus. Understanding the importance and disease specificity of these interactions could provide new therapeutic options.

Evaluation of FGFR targeting in breast cancer through interrogation of patient-derived models

BackgroundParticular breast cancer subtypes pose a clinical challenge due to limited targeted therapeutic options and/or poor responses to the existing targeted therapies. While cell lines provide useful pre-clinical models, patient-derived xenografts (PDX) and organoids (PDO) provide significant advantages, including maintenance of genetic and phenotypic heterogeneity, 3D architecture and for PDX, tumor–stroma interactions. In this study, we applied an integrated multi-omic approach across panels of breast cancer PDXs and PDOs in order to identify candidate therapeutic targets, with a major focus on specific FGFRs.MethodsMS-based phosphoproteomics, RNAseq, WES and Western blotting were used to characterize aberrantly activated protein kinases and effects of specific FGFR inhibitors. PDX and PDO were treated with the selective tyrosine kinase inhibitors AZD4547 (FGFR1-3) and BLU9931 (FGFR4). FGFR4 expression in cancer tissue samples and PDOs was assessed by immunohistochemistry. METABRIC and TCGA datasets were interrogated to identify specific FGFR alterations and their association with breast cancer subtype and patient survival.ResultsPhosphoproteomic profiling across 18 triple-negative breast cancers (TNBC) and 1 luminal B PDX revealed considerable heterogeneity in kinase activation, but 1/3 of PDX exhibited enhanced phosphorylation of FGFR1, FGFR2 or FGFR4. One TNBC PDX with high FGFR2 activation was exquisitely sensitive to AZD4547. Integrated ‘omic analysis revealed a novel FGFR2-SKI fusion that comprised the majority of FGFR2 joined to the C-terminal region of SKI containing the coiled-coil domains. High FGFR4 phosphorylation characterized a luminal B PDX model and treatment with BLU9931 significantly decreased tumor growth. Phosphoproteomic and transcriptomic analyses confirmed on-target action of the two anti-FGFR drugs and also revealed novel effects on the spliceosome, metabolism and extracellular matrix (AZD4547) and RIG-I-like and NOD-like receptor signaling (BLU9931). Interrogation of public datasets revealed FGFR2 amplification, fusion or mutation in TNBC and other breast cancer subtypes, while FGFR4 overexpression and amplification occurred in all breast cancer subtypes and were associated with poor prognosis. Characterization of a PDO panel identified a luminal A PDO with high FGFR4 expression that was sensitive to BLU9931 treatment, further highlighting FGFR4 as a potential therapeutic target.ConclusionsThis work highlights how patient-derived models of human breast cancer provide powerful platforms for therapeutic target identification and analysis of drug action, and also the potential of specific FGFRs, including FGFR4, as targets for precision treatment.

3D Tumor Models for Breast Cancer: Whither We Are and What We Need.

Three-dimensional (3D) models have led to a paradigm shift in disease modeling in vitro, particularly for cancer. The past decade has seen a phenomenal increase in the development of 3D models for various types of cancers with a focus on studying stemness, invasive behavior, angiogenesis, and chemoresistance of cancer cells, as well as contributions of its stroma, which has expanded our understanding of these processes. Cancer biology is moving into exploring the emerging hallmarks of cancer, such as inflammation, immune evasion, and reprogramming of energy metabolism. Studies into these emerging concepts have provided novel targets and treatment options such as antitumor immunotherapy. However, 3D models that can investigate the emerging hallmarks are few and underexplored. As commonly used immunocompromised mice and syngenic mice cannot accurately mimic human immunology, stromal interactions, and metabolism and require the use of prohibitively expensive humanized mice, there is tremendous scope to develop authentic 3D tumor models in these areas. Taking the specific case of breast cancer, we discuss the currently available 3D models, their applications to mimic signaling in cancer, tumor-stroma interactions, drug responses, and assessment of drug delivery systems and therapies. We discuss the lacunae in the development of 3D tumor models for the emerging hallmarks of cancer, for lesser-explored forms of breast cancer, and provide insights to develop such models. We discuss how the next generation of 3D models can provide a better mimic of human cancer modeling compared to xenograft models and the scope toward preclinical models and precision medicine.

Role of Oncogenic Pathways on the Cancer Immunosuppressive Microenvironment and Its Clinical Implications in Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma is known to become resistant to treatments easily by mutations in genes involved in the key cellular pathways targeted by current molecular targeted agents (MTAs). However, the immune checkpoint inhibitor (ICI) is a promising modality for cancer treatment, in which the cancer cells are made recognizable by the immune system. Blockade of the PD1/PDL1 proteins, which help cancers evade the immune system, is currently being tested in clinical trials in combination with MTAs. In this review, several cellular signaling pathways that can alter the immune processes within the tumor and can subsequently affect the patient’s response to ICIs are detailed. This review may help scientists and clinicians to better understand the molecular factors that can influence ICI-based therapy and will help in identifying suitable cases for this type of treatment.The tumor immune microenvironment, including hepatocellular carcinoma (HCC), is complex, consisting of crosstalk among tumor components such as the cancer cells, stromal cells and immune cells. It is conceivable that phenotypic changes in cancer cells by genetic and epigenetic alterations affect the cancer–stroma interaction and anti-cancer immunity through the expression of immune checkpoint molecules, growth factors, cytokines, chemokines and metabolites that may act on the immune system in tumors. Therefore, predicting the outcome of ICI therapy requires a thorough understanding of the oncogenic signaling pathways in cancer and how they affect tumor immune evasion. In this review, we have detailed how oncogenic signaling pathways can play a role in altering the condition of the cellular components of the tumor immune microenvironment such as tumor-associated macrophages, regulatory T cells and myeloid-derived suppressor cells. The RAS/MAPK, PI3K/Akt, Wnt/β-catenin and JAK/STAT pathways have all been implicated in anti-tumor immunity. We also found that factors that reflect the immune microenvironment of the tumor, including the status of oncogenic pathways such as the volume of tumor-infiltrating T cells, expression of the immune checkpoint protein PD-1 and its ligand PD-L1, and activation of the Wnt/β-catenin signaling pathway, predict a response to ICI therapy in HCC cases.

The Immune Landscape of Osteosarcoma: Implications for Prognosis and Treatment Response.

Osteosarcoma (OS) is a high-grade malignant stromal tumor composed of mesenchymal cells producing osteoid and immature bone, with a peak of incidence in the second decade of life. Hence, although relatively rare, the social impact of this neoplasm is particularly relevant. Differently from carcinomas, molecular genetics and the role of the tumor microenvironment in the development and progression of OS are mainly unknown. Indeed, while the tumor microenvironment has been widely studied in other solid tumor types and its contribution to tumor progression has been definitely established, tumor–stroma interaction in OS has been quite neglected for years. Only recently have new insights been gained, also thanks to the availability of new technologies and bioinformatics tools. A better understanding of the cross-talk between the bone microenvironment, including immune and stromal cells, and OS will be key not only for a deeper knowledge of osteosarcoma pathophysiology, but also for the development of novel therapeutic strategies. In this review, we summarize the current knowledge about the tumor microenvironment in OS, mainly focusing on immune cells, discussing their role and implication for disease prognosis and treatment response.

SOX11, CD70, and Treg cells configure the tumor immune microenvironment of aggressive mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is a mature B-cell neoplasm with a heterogeneous clinical and biological behavior. SOX11 oncogenic expression contributes to the aggressiveness of these tumors by different mechanisms, including tumor and stromal cell interactions. However, the precise composition of the immune cell microenvironment of MCL, its possible relationship to SOX11 expression, and how it may contribute to tumor behavior is not well known. Here, we performed an integrative transcriptome analysis of 730 immune-related genes combined with the immune cell phenotype analysis by immunohistochemistry in SOX11+ and SOX11− primary nodal MCL cases and non-neoplastic reactive lymph nodes. SOX11+ MCL had a significant lower T-cell intratumoral infiltration compared with negative cases. A reduced expression of MHCI/II-like and T-cell costimulation and signaling activation related transcripts was significantly associated with poor clinical outcome. Moreover, we identified CD70 as a SOX11 direct target gene, whose overexpression was induced in SOX11+, but not SOX11− tumor cells by CD40L in vitro. CD70 was overexpressed in primary SOX11+ MCL and it was associated with an immune unbalance of the tumor microenvironment characterized by increased number of effector regulatory T (Treg) cell infiltration, higher proliferation, and aggressive clinical course. CD27 was expressed with moderate to strong intensity in 76% of cases. Overall, our results suggest that SOX11 expression in MCL is associated with an immunosuppressive microenvironment characterized by CD70 overexpression in tumor cells, increased Treg cell infiltration and downmodulation of antigen processing, and presentation and T-cell activation that could promote MCL progression and represent a potential target for tailored therapies.