Protocol to study immunodynamics in the tumor microenvironment using a tyramide signal amplification-based immunofluorescent multiplex panel.

Immunodynamics in the tumor microenvironment can be precisely examined by using multiple antigen identification approaches. Here, we present a protocol for capturing expression levels of multiple target proteins in the same specimen at single-cell resolution using a tyramide signal amplification-based immunofluorescent multiplexing system. We describe steps for tumor tissue microarray preparation, multiplex immunohistochemistry staining, image acquisition, and quantification. This protocol can quantify immune cells in tissues from patients or experimental disease models at a protein level. For complete details on the use and execution of this protocol, please refer to Chung et al. (2023),1 Tang et al. (2022),2 and Tang et al. (2022).3.

Hematopoietic Transcription Factor RUNX1 is Essential for Promoting Macrophage-Myofibroblast Transition in Non-Small-Cell Lung Carcinoma.

Macrophage-myofibroblast transition (MMT) is a newly discovered pathway for mass production of pro-tumoral cancer-associated fibroblasts (CAFs) in non-small cell lung carcinoma (NSCLC) in a TGF-ß1/Smad3 dependent manner. Better understanding its regulatory signaling in tumor microenvironment (TME) may identify druggable target for the development of precision medicine. Here, by dissecting the transcriptome dynamics of tumor-associated macrophage at single-cell resolution, a crucial role of a hematopoietic transcription factor Runx1 in MMT formation is revealed. Surprisingly, integrative bioinformatic analysis uncovers Runx1 as a key regulator in the downstream of MMT-specific TGF-ß1/Smad3 signaling. Stromal Runx1 level positively correlates with the MMT-derived CAF abundance and mortality in NSCLC patients. Mechanistically, macrophage-specific Runx1 promotes the transcription of genes related to CAF signatures in MMT cells at genomic level. Importantly, macrophage-specific genetic deletion and systemic pharmacological inhibition of TGF-ß1/Smad3/Runx1 signaling effectively prevent MMT-driven CAF and tumor formation in vitro and in vivo, representing a potential therapeutic target for clinical NSCLC.

Smad3 is essential for polarization of tumor-associated neutrophils in non-small cell lung carcinoma.

Neutrophils are dynamic with their phenotype and function shaped by the microenvironment, such as the N1 antitumor and N2 pro-tumor states within the tumor microenvironment (TME), but its regulation remains undefined. Here we examine TGF-ß1/Smad3 signaling in tumor-associated neutrophils (TANs) in non-small cell lung carcinoma (NSCLC) patients. Smad3 activation in N2 TANs is negatively correlate with the N1 population and patient survival. In experimental lung carcinoma, TANs switch from a predominant N2 state in wild-type mice to an N1 state in Smad3-KO mice which associate with enhanced neutrophil infiltration and tumor regression. Neutrophil depletion abrogates the N1 anticancer phenotype in Smad3-KO mice, while adoptive transfer of Smad3-KO neutrophils reproduces this protective effect in wild-type mice. Single-cell analysis uncovers a TAN subset showing a mature N1 phenotype in Smad3-KO TME, whereas wild-type TANs mainly retain an immature N2 state due to Smad3. Mechanistically, TME-induced Smad3 target genes related to cell fate determination to preserve the N2 state of TAN. Importantly, genetic deletion and pharmaceutical inhibition of Smad3 enhance the anticancer capacity of neutrophils against NSCLC via promoting their N1 maturation. Thus, our work suggests that Smad3 signaling in neutrophils may represent a therapeutic target for cancer immunotherapy.

Single-cell RNA sequencing uncovers a neuron-like macrophage subset associated with cancer pain.

Tumor innervation is a common phenomenon with unknown mechanism. Here, we discovered a direct mechanism of tumor-associated macrophage (TAM) for promoting de novo neurogenesis via a subset showing neuronal phenotypes and pain receptor expression associated with cancer-driven nocifensive behaviors. This subset is rich in lung adenocarcinoma associated with poorer prognosis. By elucidating the transcriptome dynamics of TAM with single-cell resolution, we discovered a phenomenon "macrophage to neuron-like cell transition" (MNT) for directly promoting tumoral neurogenesis, evidenced by macrophage depletion and fate-mapping study in lung carcinoma models. Encouragingly, we detected neuronal phenotypes and activities of the bone marrow-derived MNT cells (MNTs) in vitro. Adoptive transfer of MNTs into NOD/SCID mice markedly enhanced their cancer-associated nocifensive behaviors. We identified macrophage-specific Smad3 as a pivotal regulator for promoting MNT at the genomic level; its disruption effectively blocked the tumor innervation and cancer-dependent nocifensive behaviors in vivo. Thus, MNT may represent a precision therapeutic target for cancer pain.

TGF-ß signaling networks in the tumor microenvironment.

Transforming growth factor-ß (TGF-ß) signaling shows important roles in both physiology and pathology, especially in the progression of inflammatory diseases including cancer. Interestingly, TGF-ß was first reported as a cancer suppressor, but increasing evidence confirmed its protumoral actions. Paradoxically, TGF-ß can be produced by both cancer cells and stromal cells as a signaling network, which actively shapes the tumor microenvironment (TME). Surprisingly, disruption of TGF-ß signaling results in both anti-cancer and pro-tumoral phenotypes in experimental cancer models, revealing the unexpected complexity of its downstream pathways for mediating cancer progression. Thus, a better understanding of the underlying mechanisms of TGF-ß signaling at the molecular level can bring new insights for developing medications that can precisely separate the anti-cancer actions from the tumor-promoting outcomes. Here, we systematically summarized the latest discoveries of TGF-ß signaling in cancer cells and the TME and discussed their translational implications for cancer.

LncRNA-Dependent Mechanisms of Transforming Growth Factor-ß: From Tissue Fibrosis to Cancer Progression.

Transforming growth factor-ß (TGF-ß) is a crucial pathogenic mediator of inflammatory diseases. In tissue fibrosis, TGF-ß regulates the pathogenic activity of infiltrated immunocytes and promotes extracellular matrix production via de novo myofibroblast generation and kidney cell activation. In cancer, TGF-ß promotes cancer invasion and metastasis by enhancing the stemness and epithelial mesenchymal transition of cancer cells. However, TGF-ß is highly pleiotropic in both tissue fibrosis and cancers, and thus, direct targeting of TGF-ß may also block its protective anti-inflammatory and tumor-suppressive effects, resulting in undesirable outcomes. Increasing evidence suggests the involvement of long non-coding RNAs (lncRNAs) in TGF-ß-driven tissue fibrosis and cancer progression with a high cell-type and disease specificity, serving as an ideal target for therapeutic development. In this review, the mechanism and translational potential of TGF-ß-associated lncRNAs in tissue fibrosis and cancer will be discussed.

Smad3 Promotes Cancer-Associated Fibroblasts Generation via Macrophage-Myofibroblast Transition.

Cancer-associated fibroblasts (CAFs) are important in tumor microenvironment (TME) driven cancer progression. However, CAFs are heterogeneous and still largely underdefined, better understanding their origins will identify new therapeutic strategies for cancer. Here, the authors discovered a new role of macrophage-myofibroblast transition (MMT) in cancer for de novo generating protumoral CAFs by resolving the transcriptome dynamics of tumor-associated macrophages (TAM) with single-cell resolution. MMT cells (MMTs) are observed in non-small-cell lung carcinoma (NSCLC) associated with CAF abundance and patient mortality. By fate-mapping study, RNA velocity, and pseudotime analysis, existence of novel macrophage-lineage-derived CAF subset in the TME of Lewis lung carcinoma (LLC) model is confirmed, which is directly transited via MMT from M2-TAM in vivo and bone-marrow-derived macrophages (BMDM) in vitro. Adoptive transfer of BMDM-derived MMTs markedly promote CAF formation in LLC-bearing mice. Mechanistically, a Smad3-centric regulatory network is upregulated in the MMTs of NSCLC, where chromatin immunoprecipitation sequencing(ChIP-seq) detects a significant enrichment of Smad3 binding on fibroblast differentiation genes in the macrophage-lineage cells in LLC-tumor. More importantly, macrophage-specific deletion and pharmaceutical inhibition of Smad3 effectively block MMT, therefore, suppressing the CAF formation and cancer progression in vivo. Thus, MMT may represent a novel therapeutic target of CAF for cancer immunotherapy.

USMB-shMincle: a virus-free gene therapy for blocking M1/M2 polarization of tumor-associated macrophages.

Mincle is essential for tumor-associated macrophage (TAM)-driven cancer progression and represents a potential immunotherapeutic target for cancer. Nevertheless, the lack of a specific inhibitor has largely limited its clinical translation. Here, we successfully developed a gene therapeutic strategy for silencing Mincle in a virus-free and tumor-specific manner by combining RNA interference technology with an ultrasound-microbubble-mediated gene transfer system (USMB). We identified a small hairpin RNA (shRNA) sequence shMincle that can silence not only Mincle expression but also the protumoral effector production in mouse bone marrow- and human THP-1-derived macrophages in the cancer setting in vitro. By using our well-established USMB system (USMB-shMincle), the shMincle-expressing plasmids were delivered in a tissue-specific manner into xenografts of human lung carcinoma A549 and melanoma A375 in vivo. Encouragingly, we found that USMB-shMincle effectively inhibited the protumoral phenotypes of TAMs as well as the progression of both A549 and A375 xenografts in a dose-dependent manner in mice without significant side effects. Mechanistically, we identified that USMB-shMincle markedly enhanced the anticancer M1 phenotype of TAMs in the A549 and A375 xenografts by blocking the protumoral Mincle/Syk/nuclear factor κB (NF-κB) signaling axis. Thus, USMB-shMincle may represent a clinically translatable novel and safe gene therapeutic approach for cancer treatment.

AANG: A natural compound formula for overcoming multidrug resistance via synergistic rebalancing the TGF-ß/Smad signalling in hepatocellular carcinoma.

Cancer cells are high in heterogeneity and versatility, which can easily adapt to the external stresses via both primary and secondary resistance. Targeting of tumour microenvironment (TME) is a new approach and an ideal therapeutic strategy especially for the multidrug resistant cancer. Recently, we invented AANG, a natural compound formula containing traditional Chinese medicine (TCM) derived Smad3 inhibitor Naringenin (NG) and Smad7 activator Asiatic Acid (AA), for rebalancing TGF-ß/Smad signalling in the TME, and its implication on the multidrug resistance is still unexplored. Here, we observed that an equilibrium shift of the Smad signalling in patients with hepatocellular carcinoma (HCC), which was dramatically enhanced in the recurrent cases showing p-glycoprotein overexpression. We optimized the formula ratio and dosage of AANG that effectively inhibit the proliferation of our unique human multidrug resistant subclone R-HepG2. Mechanistically, we found that AANG not only inhibits Smad3 at post-transcriptional level, but also upregulates Smad7 at transcriptional level in a synergistic manner in vitro. More importantly, AANG markedly suppressed the growth and p-glycoprotein expression of R-HepG2 xenografts in vivo. Thus, AANG may represent a novel and safe TCM-derived natural compound formula for overcoming HCC with p-glycoprotein-mediated multidrug resistance.

TGF-ß Signaling: From Tissue Fibrosis to Tumor Microenvironment.

Transforming growth factor-ß (TGF-ß) signaling triggers diverse biological actions in inflammatory diseases. In tissue fibrosis, it acts as a key pathogenic regulator for promoting immunoregulation via controlling the activation, proliferation, and apoptosis of immunocytes. In cancer, it plays a critical role in tumor microenvironment (TME) for accelerating invasion, metastasis, angiogenesis, and immunosuppression. Increasing evidence suggest a pleiotropic nature of TGF-ß signaling as a critical pathway for generating fibrotic TME, which contains numerous cancer-associated fibroblasts (CAFs), extracellular matrix proteins, and remodeling enzymes. Its pathogenic roles and working mechanisms in tumorigenesis are still largely unclear. Importantly, recent studies successfully demonstrated the clinical implications of fibrotic TME in cancer. This review systematically summarized the latest updates and discoveries of TGF-ß signaling in the fibrotic TME.

TGF-ß1 Signaling: Immune Dynamics of Chronic Kidney Diseases.

Chronic kidney disease (CKD) is a major cause of morbidity and mortality worldwide, imposing a great burden on the healthcare system. Regrettably, effective CKD therapeutic strategies are yet available due to their elusive pathogenic mechanisms. CKD is featured by progressive inflammation and fibrosis associated with immune cell dysfunction, leading to the formation of an inflammatory microenvironment, which ultimately exacerbating renal fibrosis. Transforming growth factor ß1 (TGF-ß1) is an indispensable immunoregulator promoting CKD progression by controlling the activation, proliferation, and apoptosis of immunocytes via both canonical and non-canonical pathways. More importantly, recent studies have uncovered a new mechanism of TGF-ß1 for de novo generation of myofibroblast via macrophage-myofibroblast transition (MMT). This review will update the versatile roles of TGF-ß signaling in the dynamics of renal immunity, a better understanding may facilitate the discovery of novel therapeutic strategies against CKD.

Neural transcription factor Pou4f1 promotes renal fibrosis via macrophage-myofibroblast transition.

Unresolved inflammation can lead to tissue fibrosis and impaired organ function. Macrophage-myofibroblast transition (MMT) is one newly identified mechanism by which ongoing chronic inflammation causes progressive fibrosis in different forms of kidney disease. However, the mechanisms underlying MMT are still largely unknown. Here, we discovered a brain-specific homeobox/POU domain protein Pou4f1 (Brn3a) as a specific regulator of MMT. Interestingly, we found that Pou4f1 is highly expressed by macrophages undergoing MMT in sites of fibrosis in human and experimental kidney disease, identified by coexpression of the myofibroblast marker, α-SMA. Unexpectedly, Pou4f1 expression peaked in the early stage in renal fibrogenesis in vivo and during MMT of bone marrow-derived macrophages (BMDMs) in vitro. Mechanistically, chromatin immunoprecipitation (ChIP) assay identified that Pou4f1 is a Smad3 target and the key downstream regulator of MMT, while microarray analysis defined a Pou4f1-dependent fibrogenic gene network for promoting TGF-ß1/Smad3-driven MMT in BMDMs at the transcriptional level. More importantly, using two mouse models of progressive renal interstitial fibrosis featuring the MMT process, we demonstrated that adoptive transfer of TGF-ß1-stimulated BMDMs restored both MMT and renal fibrosis in macrophage-depleted mice, which was prevented by silencing Pou4f1 in transferred BMDMs. These findings establish a role for Pou4f1 in MMT and renal fibrosis and suggest that Pou4f1 may be a therapeutic target for chronic kidney disease with progressive renal fibrosis.

The Emerging Role of Innate Immunity in Chronic Kidney Diseases.

Renal fibrosis is a common fate of chronic kidney diseases. Emerging studies suggest that unsolved inflammation will progressively transit into tissue fibrosis that finally results in an irreversible end-stage renal disease (ESRD). Renal inflammation recruits and activates immunocytes, which largely promotes tissue scarring of the diseased kidney. Importantly, studies have suggested a crucial role of innate immunity in the pathologic basis of kidney diseases. This review provides an update of both clinical and experimental information, focused on how innate immune signaling contributes to renal fibrogenesis. A better understanding of the underlying mechanisms may uncover a novel therapeutic strategy for ESRD.

LRNA9884, a Novel Smad3-Dependent Long Noncoding RNA, Promotes Diabetic Kidney Injury in db/db Mice via Enhancing MCP-1-Dependent Renal Inflammation.

Transforming growth factor-ß/Smad3 signaling plays an important role in diabetic nephropathy, but its underlying working mechanism remains largely unexplored. The current study uncovered the pathogenic role and underlying mechanism of a novel Smad3-dependent long noncoding RNA (lncRNA) (LRNA9884) in type 2 diabetic nephropathy (T2DN). We found that LRNA9884 was significantly upregulated in the diabetic kidney of db/db mice at the age of 8 weeks preceding the onset of microalbuminuria and was associated with the progression of diabetic renal injury. LRNA9884 was induced by advanced glycation end products and tightly regulated by Smad3, and its levels were significantly blunted in db/db mice and cells lacking Smad3. More importantly, kidney-specific silencing of LRNA9884 effectively attenuated diabetic kidney injury in db/db mice, as shown by the reduction of histological injury, albuminuria excretion, and serum creatinine. Mechanistically, we identified that LRNA9884 promoted renal inflammation-driven T2DN by triggering MCP-1 production at the transcriptional level, and its direct binding significantly enhanced the promoter activity of MCP-1. Thus, LRNA9884 is a novel Smad3-dependent lncRNA that is highly expressed in db/db mice associated with T2DN development. Targeting of LRNA9884 effectively blocked MCP-1-dependent renal inflammation, therefore suppressing the progressive diabetic renal injury in db/db mice. This study reveals that LRNA9884 may be a novel and precision therapeutic target for T2DN in the future.

Transforming growth factor-ß signalling in renal fibrosis: from Smads to non-coding RNAs.

Transforming growth factor-ß (TGF-ß) is the key player in tissue fibrosis. However, antifibrotic therapy targeting this multifunctional protein may interfere with other physiological processes to cause side effects. Thus, precise therapeutic targets need to be identified by further understanding the underlying mechanisms of TGF-ß1 signalling during fibrogenesis. Equilibrium of Smad signalling is crucial for TGF-ß-mediated renal fibrosis, where Smad3 is pathogenic but Smad2 and Smad7 are protective. The activation of TGF-ß1/Smad signalling triggers extracellular matrix deposition, and local myofibroblast generation and activation. Mechanistic studies have shown that TGF-ß/Smad3 transits the microRNA profile from antifibrotic to profibrotic and therefore promotes renal fibrosis via regulating non-coding RNAs at transcriptional levels. More importantly, disease-specific Smad3-dependent long non-coding RNAs have been recently uncovered from mouse kidney disease models and may represent novel precision therapeutic targets for chronic kidney disease. In this review, mechanisms of TGF-ß-driven renal fibrosis via non-coding RNAs and their translational capacities will be discussed in detail.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

Dietary and Supplemental Vitamin C and D on Symptom Severity and Physical Function in Knee Osteoarthritis.

Vitamins C and D have been associated with decreasing pain and increasing function but these associations are not definitive. This cross-sectional study explores what relationships supplemental and dietary intake of vitamins C and D have on pain severity and physical function in patients with knee osteoarthritis. Using data from the Osteoarthritis Initiative, we performed regression analyses to examine relationships between vitamins C and D, pain, and function. Dietary vitamin D and dietary vitamin C were divided into >90th, 50th-90th, and <50th percentile. The high percentile group for supplemental vitamin D was divided into >85th percentile, whereas the high percentile group for supplemental vitamin C was divided into >90th percentile. We found the 90th/85th percentile levels of dietary and supplemental vitamin D to be positively associated with pain (ß = 0.180; p = 0.028) and inversely related to physical function (ß = -0.150, p = 0.028), respectively. Daily intake of vitamin C showed no statistical significance. We found that supplementary vitamin D was strongly associated with lessened disability for knee OA patients. The unexpected finding that associated dietary vitamin D with greater knee pain merits further study.

TGF-ß1 signaling in kidney disease: From Smads to long non-coding RNAs.

Transforming growth factor-ß1 (TGF-ß1) has an essential role in the development of kidney diseases. However, targeting TGF-ß1 is not a good strategy for fibrotic diseases due to its multifunctional characteristic in physiology. A precise therapeutic target maybe identified by further resolving the underlying TGF-ß1 driven mechanisms in renal inflammation and fibrosis. Smad signaling is uncovered as a key pathway of TGF-ß1-mediated renal injury, where Smad3 is hyper-activated but Smad7 is suppressed. Mechanistic studies revealed that TGF-ß1/Smad3 is capable of promoting renal inflammation and fibrosis via regulating non-coding RNAs. More importantly, involvement of disease- and tissue-specific TGF-ß1-dependent long non-coding RNAs (lncRNA) have been recently recognized in a number of kidney diseases. In this review, current understanding of TGF-ß1 driven lncRNAs in the pathogenesis of kidney injury, diabetic nephropathy and renal cell carcinoma will be intensively discussed.

Inhibition of human equilibrative nucleoside transporters by 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine.

Equilibrative nucleoside transporters (ENTs) play a crucial role in the transport of nucleoside and nucleoside analogues, which are important for nucleotide synthesis and chemotherapy. In addition, ENTs regulate extracellular adenosine levels in the vicinity of its receptors and hence influence adenosine-related functions. The clinical applications of ENT inhibitors in the treatment of cardiovascular diseases and cancer therapy have been explored in numerous studies. However, all ENT inhibitors to date are selective for ENT1 but not ENT2. In the present study, we investigated the novel compound 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) as an inhibitor of ENT1 and ENT2. Nucleoside transporter-deficient PK15NTD cells stably expressing ENT1 and ENT2 showed that FPMINT inhibited [3H]uridine and [3H]adenosine transport through both ENT1 and ENT2 in a concentration-dependent manner. The IC50 value of FPMINT for ENT2 was 5-10-fold less than for ENT1, and FPMINT could not be displaced with excess washing. Kinetic studies revealed that FPMINT reduced Vmax of [3H]uridine transport in ENT1 and ENT2 without affecting KM. Therefore, we conclude that FPMINT inhibits ENTs in an irreversible and non-competitive manner. Although already selective for ENT2 over ENT1, further modification of the chemical structure of FPMINT may lead to even better ENT2-selective inhibitors of potential clinical, physiological and pharmacological importance.