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Immunoperoxidase (a-c) and immunofluorescence (d-e) detection in osteoclast rich areas and surrounding stroma (f and g), and clinicopathological correlations of Cx43 protein levels (h and i) in giant cell tumor of bone. Examples of tumors with moderate (a; score 3) and high (b; score 8) Cx43 levels in mononuclear cells. Strong Cx43 reaction in the preexisting osteoblast layer around bone spicules and in osteocytes (arrowhead) (c). A tumor nest and adjacent ring of reactive stroma are annotated separately for counting Cx43 (Alexa564, red) plaques (d; OC-osteoclasts). Higher power of (d) with osteoclasts encircled (e). Digital image segmentation highlights Cx43 plaques in orange for automated counting (f). Both the Cx43 positive area fraction (g) and the number of Cx43 positive plaques (h) are significantly reduced within tumor nests (p<0.01). Cx43 levels are also significantly reduced in aggressive vs active and in aggressive vs latent clinicoradiological tumor stages (i). Scale bar on (a) represents 30 μm on a, b and c; 80 μm on d, 30 μm on e and 15 μm on f.
Source publication
Missense mutations of the GJA1 gene encoding the gap junction channel protein connexin43 (Cx43) cause bone malformations resulting in oculodentodigital dysplasia (ODDD), while GJA1 null and ODDD mutant mice develop osteopenia. In this study we investigated Cx43 expression and channel functions in giant cell tumor of bone (GCTB), a locally aggressiv...
Citations
... 36 The gap junction inhibitor 1-heptanol 9 also failed to inhibit RNA acquisition ( Figure 3E), but did significantly reduce transfer of Calcein dye between COCA KCs ( Figure S3J), which is partially mediated by gap junctions. 37 Our findings were not limited to RNA acquisition from KCs by MutuDC1s. We observed roughly similar responses with MutuDC1s or primary splenic DCs combined with an unrelated cancer cell line, B16 ( Figures S3K and S3L). ...
In a previous report, keratinocytes were shown to share their gene expression profile with surrounding Langerhans cells (LCs), influencing LC biology. Here, we investigated whether transferred material could substitute for lost gene products in cells subjected to Cre/Lox conditional gene deletion. We found that in human Langerin-Cre mice, epidermal LCs and CD11b+CD103+ mesenteric DCs overcome gene deletion if the deleted gene was expressed by neighboring cells. The mechanism of material transfer differed from traditional antigen uptake routes, relying on calcium and PI3K, being susceptible to polyguanylic acid inhibition, and remaining unaffected by inflammation. Termed intracellular monitoring, this process was specific to DCs, occurring in all murine DC subsets tested and human monocyte-derived DCs. The transferred material was presented on MHC-I and MHC-II, suggesting a role in regulating immune responses.
... For the identification of primary stromal cells, we performed a qRT-PCR assay to test the expression of CD34 and CD163, which are commonly used markers for GCTB [18,19]. Additionally, we carried out the Western blot to quantify the CSF1R protein level, which is abnormally expressed in giant cell tumors [20]. ...
Background: Giant cell tumor of bone (GCTB) is a locally aggressive bone tumour aggravated by stromal cell proliferation and metastasis.
Objective: We investigated the mechanism of action of human chorionic gonadotropin (HCG) in mediating GCTB proliferation and invasion.
Methods: The expression of HCG was quantified using quantitative real-time PCR. After the primary stromal cells were isolated and identified, the function of HCG in GCTB was estimated using the cell counting kit-8, flow cytometry, scratch experiment, transwell assay, Western blot, and immunofluorescence. Moreover, the mechanism of HCG was assessed through western blotting.
Results: HCG expression was decreased in clinical tissue samples from patients with GCTB. We validated that HCG repressed stromal cell proliferation, migration, invasion, autophagy, and epithelial-mesenchymal transition (EMT) and promoted cell apoptosis in GCTB. We also verified that HCG repressed the autophagy and EMT of stromal cells through the Smad signaling axis in GCTB. HCG inhibited the transduction of the Smad signaling pathway by restraining the binding of the TGF-β II receptor to ligand Activin A.
Conclusion: HCG restrained the Smad signaling pathway by antagonizing TGF-β signaling in GCTB. HCG may serve as a useful patent to treat GCTB.
... ; https://doi.org/10.1101/2023.07.22.550169 doi: bioRxiv preprint significantly reduce transfer of Calcein dye between COCA KCs (Supplemental Fig. 3H), which is partially mediated by gap junctions (36). Our findings were not limited to RNA acquisition from KCs. ...
Macrophages and dendritic cells (DCs) in peripheral tissue interact closely with their local microenvironment by scavenging protein and nucleic acids released by neighboring cells. Material transfer between cell types is necessary for pathogen detection and antigen presentation, but thought to be relatively limited in scale. Recent reports, however, demonstrate that the quantity of transferred material can be quite large when DCs are in direct contact with live cells. This observation may be problematic for conditional gene deletion models that assume gene products will remain in the cell they are produced in. Here, we investigate whether conditional gene deletions induced by the widely used Cre/Lox system can be overcome at the protein level in DCs. Of concern, using the human Langerin Cre mouse model, we find that epidermal Langerhans cells and CD11b+CD103+ mesenteric DCs can overcome gene deletion if the deleted gene is expressed by neighboring cells. Surprisingly, we also find that the mechanism of material transfer does not resemble known mechanisms of antigen uptake, is dependent on extra- and intracellular calcium, PI3K, and scavenger receptors, and mediates a majority of material transfer to DCs. We term this novel process intracellular monitoring, and find that it is specific to DCs, but occurs in all murine DC subsets tested, as well as in human DCs. Transferred material is successfully presented and cross presented on MHC-II and MHC-I, and occurs between allogeneic donor and acceptors cells—implicating this widespread and unique process in immunosurveillance and organ transplantation.
ONE SENTENCE SUMMARY
Dendritic cells maintain RNA and protein levels for conditionally deleted genes by siphoning material from neighboring cells using a novel mechanism.
... Te patients were classifed into high-risk or low-risk groups according to the Rad-score, whose threshold was identifed by using the X-tile. In addition, univariate Cox regression analysis was used for other risk features highly correlated with survival, such as CT semantics, clinicopathologic, and TIME parameters [16,17]. Finally, we combined the above-selected risk features with the Rad-score and used the backward selection method [18] to incorporate the abovementioned risk factors into the multivariate Cox regression model. ...
To improve prognosis of cancer patients and determine the integrative value for analysis of disease-free survival prediction, a clinic investigation was performed involving with 146 non-small cell lung cancer (NSCLC) patients (83 men and 73 women; mean age: 60.24 years ± 8.637) with a history of surgery. Teir computed tomography (CT) radiomics, clinical records, and tumor immune features were frstly obtained and analyzed in this study. Histology and immunohistochemistry were also performed to establish a multimodal nomogram through the ftting model and cross-validation. Finally, Z test and decision curve analysis (DCA) were performed to evaluate and compare the accuracy and diference of each model. In all, seven radiomics features were selected to construct the radiomics score model. Te clinicopathological and immunological factors model, including T stage, N stage, microvascular invasion, smoking quantity, family history of cancer, and immunophenotyping. Te C-index of the comprehensive nomogram model on the training set and test set was 0.8766 and 0.8426 respectively, which was better than that of the clin-icopathological-radiomics model (Z test, P =0.041<0.05), radiomics model and clinicopathological model (Z test, P =0.013<0.05 and P =0.0097<0.05). Integrative nomogram based on computed tomography radiomics, clinical and immunophenotyping can be served as efective imaging biomarker to predict DFS of hepatocellular carcinoma after surgical resection.
... In the case of the giant-cell tumor of bone, Cx43 participates in the fusion of osteoclasts and monocytes. This abnormal cellular cooperation is most likely associated with an attempt by monocytes to regenerate the cancer-associated damage to bone tissues, which instead leads to an increase in the size of the tumor [128]. ...
Simple Summary
Connexins are proteins which comprise gap junctions in cells. These junctions can directly connect neighboring cells and the cell interior with the extracellular microenvironment and thus they act as tissue integrators. Alterations in connexin regulation can lead to unfavorable shifts in the tissue adhesive context thus eradicating the constraints of the normal tissue microenvironment, triggering (or enhancing) cell motility. This review tries to examine the role of connexins in orchestrating the tumor microenvironment and hence their role in malignancy.
Abstract
Today’s research on the processes of carcinogenesis and the vital activity of tumor tissues implies more attention be paid to constituents of the tumor microenvironment and their interactions. These interactions between cells in the tumor microenvironment can be mediated via different types of protein junctions. Connexins are one of the major contributors to intercellular communication. They form the gap junctions responsible for the transfer of ions, metabolites, peptides, miRNA, etc., between neighboring tumor cells as well as between tumor and stromal cells. Connexin hemichannels mediate purinergic signaling and bidirectional molecular transport with the extracellular environment. Additionally, connexins have been reported to localize in tumor-derived exosomes and facilitate the release of their cargo. A large body of evidence implies that the role of connexins in cancer is multifaceted. The pro- or anti-tumorigenic properties of connexins are determined by their abundance, localization, and functionality as well as their channel assembly and non-channel functions. In this review, we have summarized the data on the contribution of connexins to the formation of the tumor microenvironment and to cancer initiation and progression.
... Double immunofluorescence (NGFR plus Cx43) reactions of 10 cases of myelofibrosis were studied using semi-automated image analysis as described before [24]. Immunostained slides were digitalized at separated color channels (R, G and B for cell nuclei) through five layers at each field of views (FOVs) and analyzed at extended focus using the HistoQuant program (3DHistech). ...
In myelofibrosis, pathologically enhanced extracellular matrix production due to aberrant cytokine signalling and clonal megakaryocyte functions result( s ) in impaired hemopoiesis. Disease progression is still determined by detecting reticulin and collagen fibrosis with Gomori’s silver impregnation. Here, we tested whether the expression growth related biomarkers L-NGFR/CD271, phospho-ERK1-2 and CXCL12 can be linked to the functional activation of bone marrow stromal cells during primary myelofibrosis progression. Immunoscores for all tested biomarkers showed varying strength of positive statistical correlation with the silver impregnation based myelofibrosis grades. The intimate relationship between spindle shaped stromal cells positive for all three markers and aberrant megakaryocytes was likely to reflect their functional cooperation. L-NGFR reaction was restricted to bone marrow stromal cells and revealed the whole length of their processes. Also, L-NGFR positive cells showed the most intersections, the best statistical correlations with myelofibrosis grades and the strongest interrater agreements. CXCL12 reaction highlighted stromal cell bodies and a weak extracellular staining in line with its constitutive release. Phospho-ERK1-2 reaction showed a similar pattern to CXCL12 in stromal cells with an additional nuclear staining in agreement with its role as a transcription factor. Both p -ERK1-2 and CXCL12 were also expressed at a moderate level in sinus endothelial cells. Connexin 43 gap junction communication channels, known to be required for CXCL12 release to maintain stem cell niche, were also expressed progressively in the myelofibrotic stromal network as a support of compartmental functions. Our results suggest that, diverse growth related pathways are activated in the functionally coupled bone marrow stromal cells during myelofibrosis progression. L-NGFR expression can be a useful biological marker of stromal cell activation which deserves diagnostic consideration for complementing Gomori’s silver impregnation.
... Cnx43 participates in the fusion of osteoclasts and monocytes in the microenvironment of giant-cell tumor of bone. This abnormal cellular cooperation is most likely associated with an attempt by monocytes to regenerate the cancer-associated damage to the bone tissue, which instead leads to an increase in the size of the tumor [140]. Cnx contacts between tumor cells also mediate the escape of immune surveillance. ...
The modern paradigm of studying the processes of carcinogenesis and vital activity of tumor tissues implies increased attention to constituents of tumor microenvironment (TME) and their interactions. These interactions between the cells in TME can be mediated via protein junctions of different types. Connexins (Cnxs) are one of the major contributors to intercellular communication. They form gap junctions responsible for the transfer of ions, metabolites, peptides, miRNA, etc. between neighboring tumor cells as well as between tumor and stromal cells. Cnx hemichannels mediate purinergic signaling and bidirectional molecular transport with the extracellular environment. Additionally, Cnxs were reported to localize in tumor-derived exosomes and facilitate the release of their cargo. A large body of evidence implies that the role of connexins in cancer is multifaceted. Pro- or anti-tumorigenic properties of connexins are determined by their abundance, localization and functionality as well as channel assembly and non-channel functions. In this review we have summarized the data on the Cnxs contribution in TME and to the cancer initiation and progression.
... The proliferating, neoplastic stromal cells, generally carrying H3F3A G34W mutation [8], are the major drivers of osteoclastogenesis and pathological bone resorption [9,10]. They produce canonical (RANKL/ M-CSF) and non-canonical (e.g., LIGHT, TNFα, IL-6 or vascular endothelial growth factor-VEGF) growth factors and hypoxia inducible factors 1α and 2α [14], which can either directly or through autocrine activation promote osteoclastogenesis and osteolysis [11,12]. We have shown earlier that besides the replication activity of neoplastic stromal cells, their elevated epidermal growth factor receptor (EGFR) signaling and deregulated gap junction connexin43 expression and channel functions, can contribute to GCTB progression, mediated by GCs [4,13,14]. ...
... They produce canonical (RANKL/ M-CSF) and non-canonical (e.g., LIGHT, TNFα, IL-6 or vascular endothelial growth factor-VEGF) growth factors and hypoxia inducible factors 1α and 2α [14], which can either directly or through autocrine activation promote osteoclastogenesis and osteolysis [11,12]. We have shown earlier that besides the replication activity of neoplastic stromal cells, their elevated epidermal growth factor receptor (EGFR) signaling and deregulated gap junction connexin43 expression and channel functions, can contribute to GCTB progression, mediated by GCs [4,13,14]. ...
Cells of the monocyte macrophage lineage form multinucleated giant cells (GCs) by fusion, which may express some cell cycle markers. By using a comprehensive marker set, here we looked for potential replication activities in GCs, and investigated whether these have diagnostic or clinical relevance in giant cell tumor of bone (GCTB). GC rich regions of 10 primary and 10 first recurrence GCTB cases were tested using immunohistochemistry in tissue microarrays. The nuclear positivity rate of the general proliferation marker, replication licensing, G1/S-phase, S/G2/M-phase, mitosis promoter, and cyclin dependent kinase (CDK) inhibitor reactions was analyzed in GCs. Concerning Ki67, moderate SP6 reaction was seen in many GC nuclei, while B56 and Mib1 positivity was rare, but the latter could be linked to more aggressive (p = 0.012) phenotype. Regular MCM6 reaction, as opposed to uncommon MCM2, suggested an initial DNA unwinding. Early replication course in GCs was also supported by widely detecting CDK4 and cyclin E, for the first time, and confirming cyclin D1 upregulation. However, post-G1-phase markers CDK2, cyclin A, geminin, topoisomerase-2a, aurora kinase A, and phospho-histone H3 were rare or missing. These were likely silenced by upregulated CDK inhibitors p15INK4b, p16INK4a, p27KIP1, p53 through its effector p21WAF1 and possibly cyclin G1, consistent with the prevention of DNA replication. In conclusion, the upregulation of known and several novel cell cycle progression markers detected here clearly verify early replication activities in GCs, which are controlled by cell cycle arresting CDK inhibitors at G1 phase, and support the functional maturation of GCs in GCTB.
... Connexin 43 was observed by punctate staining in the hydrogel ( Figure 2B), which is consistent with connexin 43 staining in a 2D culture control ( Figure 2C) and typical for connexin 43 (negative control images included in Figure S2, Supporting Information). [61,62] Connexin 43 comprises hemichannels and gap junctions along the cell membrane and is essential for osteocyte function, survival, and differentiation. [63][64][65] Concomitant, normalized expression (NE) of osteocyte-related genes Dmp1 and Sost, as measured by qPCR, increased over culture time ( Figure 2D). ...
Osteocytes are mechanosensitive cells that orchestrate signaling in bone and cartilage across the osteochondral unit. The mechanisms by which osteocytes regulate osteochondral homeostasis and degeneration in response to mechanical cues remain unclear. This study introduces a novel 3D hydrogel bilayer composite designed to support osteocyte differentiation and bone matrix deposition in a bone‐like layer and to recapitulate key aspects of the osteochondral unit's complex loading environment. The bilayer hydrogel is fabricated with a soft cartilage‐like layer overlaying a stiff bone‐like layer. The bone‐like layer contains a stiff 3D‐printed hydrogel structure infilled with a soft, degradable, cellular hydrogel. The IDG‐SW3 cells embedded within the soft hydrogel mature into osteocytes and produce a mineralized collagen matrix. Under dynamic compressive strains, near‐physiological levels of strain are achieved in the bone layer (≤ 0.08%), while the cartilage layer bears the majority of the strains (>99%). Under loading, the model induces an osteocyte response, measured by prostaglandin E2, that is frequency, but not strain, dependent: a finding attributed to altered fluid flow within the composite. Overall, this new hydrogel platform provides a novel approach to study osteocyte mechanobiology in vitro in an osteochondral tissue‐mimetic environment.
... Proteins were extracted using an extraction buffer made freshly by mixing 20 mM Tris, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100; supplemented with 10 mM NaF, 0.5 mM NaVO3 and 1:200 Protease Inhibitor Cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA), as previously described [57]. Briefly, cells were washed with PBS, a 150 µL extraction buffer was added and the lysate was collected with a cell scraper. ...
The poor outcome of pancreas ductal adenocarcinomas (PDAC) is frequently linked to therapy resistance. Modulated electro-hyperthermia (mEHT) generated by 13.56 MHz capacitive radiofrequency can induce direct tumor damage and promote chemo- and radiotherapy. Here, we tested the effect of mEHT either alone or in combination with radiotherapy using an in vivo model of Panc1, a KRAS and TP53 mutant, radioresistant PDAC cell line. A single mEHT shot of 60 min induced ~50% loss of viable cells and morphological signs of apoptosis including chromatin condensation, nuclear shrinkage and apoptotic bodies. Most mEHT treatment related effects exceeded those of radiotherapy, and these were further amplified after combining the two modalities. Treatment related apoptosis was confirmed by a significantly elevated number of annexin V single-positive and cleaved/activated caspase-3 positive tumor cells, as well as sub-G1-phase tumor cell fractions. mEHT and mEHT+radioterapy caused the moderate accumulation of γH2AX positive nuclear foci, indicating DNA double-strand breaks and upregulation of the cyclin dependent kinase inhibitor p21waf1 besides the downregulation of Akt signaling. A clonogenic assay revealed that both mono- and combined treatments affected the tumor progenitor/stem cell populations too. In conclusion, mEHT treatment can contribute to tumor growth inhibition and apoptosis induction and resolve radioresistance of Panc1 PDAC cells.
