MOFs-Based Nanoagents Enable Sequential Damage to Cancer-Associated Fibroblast and Tumor Cells for Phototriggered Tumor Microenvironment Regulation.

A composite nanoagent capable of phototriggered tumor microenvironment (TME) regulation is developed based on copper (II) metal-organic frameworks (MOFs) with encapsulation of blebbistatin (Bb) and surface modification of fibroblast activation protein-αtargeted peptide (Tp). Tp enables active targeting of the nanoagents to cancer-associated fibroblast (CAF) while near-infrared light triggers Cu2+ -to-Cu+ photoreduction in MOFs, which brings about the collapse of MOFs and the release of Bb and Cu+ . Bb mediates photogeneration of hydroxyl radicals (•OH) and therefore inhibits extracellular matrix production by inducing CAF apoptosis, which facilitates the penetration of nanoagent to deep tumor tissue. The dual-channel generation of •OH based on Bb and the Cu+ species, via distinct mechanisms, synergistically reinforces oxidative stress in TME capable of inducing immunogenic cell death, which activates the antitumor immune response and therefore reverses the immunosuppressive TME. The synergistic antitumor phototherapy efficacy of such a type of nanoagent based on the abovementioned TME remodeling is unequivocally verified in a cell-derived tumor xenograft model.

Bruceine D suppresses CAF-promoted TNBC metastasis under TNF-α stimulation by inhibiting Notch1-Jagged1/NF-κB(p65) signaling.

BACKGROUND: Triple-negative breast cancer (TNBC) has a poor prognosis because of its high degree of malignancy and the lack of effective treatment options. Cancer-associated fibroblasts (CAFs) comprise the most abundant stromal cells in the tumor microenvironment (TME), leading to functional impairments and facilitating tumor metastasis. Excessive TNF-α further promotes cross-talk between different cells in TME. Therefore, there is an urgent need to develop more effective therapies and potential drugs that target the key factors that promote TNBC metastasis. PURPOSE: The study aimed to evaluate the efficacy of Bruceine D, an active compound derived from the Chinese herb Brucea javanica, in inhibiting metastasis and elucidate the underlying mechanism of action in TNBC. METHODS: In vitro, the clonogenic and the Transwell assays were used to assess the effects of Bruceine D on the proliferation, migration and invasion abilities of co-cultured CAFs and MDA-MB-231 (4T1) cells under TNF-α stimulation. TNF-α, IL-6, CXCL12, TGF-ß1, and MMP9 levels in the supernatant of co-cultured cells were determined using ELISA. Western blotting was utilized to detect the expression levels of proteins related to the Notch1-Jagged1/NF-κB(p65) pathway. In vivo, the anti-tumor growth and anti-metastatic effectiveness of Bruceine D was evaluated by determining tumor weight, number of metastatic lesions, and pathological changes in the tumor and lung/liver tissues. The inhibitory effect of Bruceine D on α-SMA+ CAFs activation and CAF-medicated extracellular matrix remodeling was accessed using immunohistochemistry, immunofluorescence, and Masson and Sirius Red staining. The expression levels of Notch1, Jagged1 and p-NF-κB(p65) proteins in the primary tumors were measured by immunohistochemistry and western blotting. RESULTS: In vitro, Bruceine D significantly inhibited the migration and invasion of co-cultured CAFs and MDA-MB-231 (4T1) cells under TNF-α stimulation, reduced the expression of tumor-promoting and matrix-remodeling cytokines secreted by CAFs, and hindered the mutual activation of Notch1-Jagged1 and NF-κB(p65). In vivo, Bruceine D significantly suppressed tumor growth and the formation of lung and liver metastases by decreasing TNF-α stimulated α-SMA+ CAFs activation, collagen fibers, MMPs production, and inhibited Notch1-Jagged1/NF-κB(p65) signaling in TNBC-bearing mice. CONCLUSION: Bruceine D effectively weakened the "tumor-CAF-inflammation" network by inhibiting the mutual activation of Notch1-Jagged1 and NF-κB(p65) and thereby suppressed TNBC metastasis. This study first explored that Bruceine D disrupted the cross-talk between CAFs and tumor cells under TNF-α stimulation to inhibit the metastasis of TNBC, and highlighted the potential of Bruceine D as therapeutic agent for suppressing tumor metastasis.

Shikonin reverses cancer-associated fibroblast-induced gemcitabine resistance in pancreatic cancer cells by suppressing monocarboxylate transporter 4-mediated reverse Warburg effect.

BACKGROUND: Gemcitabine is a first-line chemotherapeutic agent for pancreatic cancer (PC); however, most patients who receive adjuvant gemcitabine rapidly develop resistance and recurrence. Cancer-associated fibroblasts (CAFs) are a crucial component of the tumor stroma that contribute to gemcitabine-resistance. There is thus an urgent need to find a novel therapeutic strategy to improve the efficacy of gemcitabine in PC cells under CAF-stimulation. PURPOSE: To investigate if shikonin potentiates the therapeutic effects of gemcitabine in PC cells with CAF-induced drug resistance. METHODS: PC cell-stimulated fibroblasts or primary CAFs derived from PC tissue were co-cultured with PC cells to evaluate the ability of shikonin to improve the chemotherapeutic effects of gemcitabine in vitro and in vivo. Glucose uptake assay, ATP content analysis, lactate measurement, real-time PCR, immunofluorescence staining, western blot, and plasmid transfection were used to investigate the underlying mechanism. RESULTS: CAFs were innately resistant to gemcitabine, but shikonin suppressed the PC cell-induced transactivation and proliferation of CAFs, reversed CAF-induced resistance, and restored the therapeutic efficacy of gemcitabine in the co-culture system. In addition, CAFs underwent a reverse Warburg effect when co-cultured with PC cells, represented by enhanced aerobic glycolytic metabolism, while shikonin reduced aerobic glycolysis in CAFs by reducing their glucose uptake, ATP concentration, lactate production and secretion, and glycolytic protein expression. Regarding the mechanism underlying these sensitizing effects, shikonin suppressed monocarboxylate transporter 4 (MCT4) expression and cellular membrane translocation to inhibit aerobic glycolysis in CAFs. Overexpression of MCT4 accordingly reversed the inhibitory effects of shikonin on PC cell-induced transactivation and aerobic glycolysis in CAFs, and reduced its sensitizing effects. Furthermore, shikonin promoted the effects of gemcitabine in reducing the growth of tumors derived from PC cells and CAF co-inoculation in BALB/C mice, with no significant systemic toxicity. CONCLUSION: These results indicate that shikonin reduced MCT4 expression and activation, resulting in inhibition of aerobic glycolysis in CAFs and overcoming CAF-induced gemcitabine resistance in PC. Shikonin is a promising chemosensitizing phytochemical agent when used in combination with gemcitabine for PC treatment. The results suggest that disrupting the metabolic coupling between cancer cells and stromal cells might provide an attractive strategy for improving gemcitabine efficacy.

Trellis tree-based analysis reveals stromal regulation of patient-derived organoid drug responses.

Patient-derived organoids (PDOs) can model personalized therapy responses; however, current screening technologies cannot reveal drug response mechanisms or how tumor microenvironment cells alter therapeutic performance. To address this, we developed a highly multiplexed mass cytometry platform to measure post-translational modification (PTM) signaling, DNA damage, cell-cycle activity, and apoptosis in >2,500 colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies at single-cell resolution. To compare patient- and microenvironment-specific drug responses in thousands of single-cell datasets, we developed "Trellis"-a highly scalable, tree-based treatment effect analysis method. Trellis single-cell screening revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is rarer, patient-specific, and aligns with cancer cell PTM signaling. We find that CAFs can regulate PDO plasticity-shifting proliferative colonic stem cells (proCSCs) to slow-cycling revival colonic stem cells (revCSCs) to protect cancer cells from chemotherapy.

Neutralizing tumor-related inflammation and reprogramming of cancer-associated fibroblasts by Curcumin in breast cancer therapy.

Tumor-associated inflammation plays a vital role in cancer progression. Among the various stromal cells, cancer-associated fibroblasts are promising targets for cancer therapy. Several reports have indicated potent anti-inflammatory effects attributed to Curcumin. This study aimed to investigate whether inhibiting the inflammatory function of cancer-associated fibroblasts (CAFs) with Curcumin can restore anticancer immune responses. CAFs were isolated from breast cancer tissues, treated with Curcumin, and co-cultured with patients' PBMCs to evaluate gene expression and cytokine production alterations. Blood and breast tumor tissue samples were obtained from 12 breast cancer patients with stage II/III invasive ductal carcinoma. Fibroblast Activation Protein (FAP) + CAFs were extracted from tumor tissue, treated with 10 µM Curcumin, and co-cultured with corresponding PBMCs. The expression of smooth muscle actin-alpha (α-SMA), Cyclooxygenase-2(COX-2), production of PGE2, and immune cell cytokines were evaluated using Real-Time PCR and ELISA, respectively. Analyzes showed that treatment with Curcumin decreased the expression of genes α-SMA and COX-2 and the production of PGE2 in CAFs. In PBMCs co-cultured with Curcumin-treated CAFs, the expression of FoxP3 decreased along with the production of TGF-ß, IL-10, and IL-4. An increase in IFN-γ production was observed that followed by increased T-bet expression. According to our results, Curcumin could reprogram the pro-tumor phenotype of CAFs and increase the anti-tumor phenotype in PBMCs. Thus, CAFs, as a component of the tumor microenvironment, are a suitable target for combination immunotherapies of breast cancer.

Polysaccharides from Lentinus edodes prevent acquired drug resistance to docetaxel in prostate cancer cells by decreasing the TGF-ß1 secretion of cancer-associated fibroblasts.

Prostate cancer is one of the most prevalent lethal diseases among men globally. In the treatment of prostate cancer, the limited therapeutic efficacy of the standard non-hormonal systemic therapy docetaxel (DTX) represents an important challenge. Cancer-associated fibroblasts (CAFs) play a crucial role in resistance to therapy because of their prevalence and functional pleiotropy in tumor environments. Our previous research revealed that MPSSS, a novel polysaccharide extracted from Lentinus edodes, could significantly attenuate the immunosuppressive function of myeloid suppressor cells and CAFs. In this study, we investigated whether MPSSS could potentiate the efficacy of DTX against prostate cancer by inhibiting CAF-induced chemoresistance and elucidated its underlying mechanisms. The sensitivity of PC-3 prostate cancer cells cultured with conditioned medium derived from CAFs (CAF-CM) to DTX was assessed. The resistance effect induced by CAF-CM was abolished when CAFs were pretreated with MPSSS. Bioinformatic analysis of datasets from the Gene Expression Omnibus database revealed the activation of the transforming growth factor ß1 (TGF-ß1) signaling pathway in DTX-resistant cells. Based on this finding, we demonstrated that treatment with the TGF-ß1 receptor inhibitor SB525334 reversed DTX resistance in CAFs, suggesting that TGF-ß1 secreted by CAFs was a crucial intermediary in the development of DTX resistance in PC3 cells. Further research revealed that MPSSS decreases the secretion of TGF-ß1 by inhibiting the JAK2/STAT3 pathway via Toll-like receptor 4 in CAFs. Overall, MPSSS might be a potential adjuvant treatment for DTX resistance in prostate cancer.

Targeting MFAP5 in cancer-associated fibroblasts sensitizes pancreatic cancer to PD-L1-based immunochemotherapy via remodeling the matrix.

Highly desmoplastic and immunosuppressive tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) contributes to tumor progression and resistance to current therapies. Clues targeting the notorious stromal environment have offered hope for improving therapeutic response whereas the underlying mechanism remains unclear. Here, we find that prognostic microfibril associated protein 5 (MFAP5) is involved in activation of cancer-associated fibroblasts (CAFs). Inhibition of MFAP5highCAFs shows synergistic effect with gemcitabine-based chemotherapy and PD-L1-based immunotherapy. Mechanistically, MFAP5 deficiency in CAFs downregulates HAS2 and CXCL10 via MFAP5/RCN2/ERK/STAT1 axis, leading to angiogenesis, hyaluronic acid (HA) and collagens deposition reduction, cytotoxic T cells infiltration, and tumor cells apoptosis. Additionally, in vivo blockade of CXCL10 with AMG487 could partially reverse the pro-tumor effect from MFAP5 overexpression in CAFs and synergize with anti-PD-L1 antibody to enhance the immunotherapeutic effect. Therefore, targeting MFAP5highCAFs might be a potential adjuvant therapy to enhance the immunochemotherapy effect in PDAC via remodeling the desmoplastic and immunosuppressive microenvironment.

Melatonin blunts the tumor-promoting effect of cancer-associated fibroblasts by reducing IL-8 expression and reversing epithelial-mesenchymal transition.

BACKGROUND: Most studies on melatonin have focused on tumor cells but have ignored the tumor microenvironment (TME), especially one of its important components, the cancer-associated fibroblasts (CAFs). Therefore, we attempted to explore the role of melatonin in TME. METHODS: We investigated the regulatory role of melatonin in the tumor-promoting effect of CAFs and its underlying mechanism by using cell and animal models. RESULTS: CAFs promoted tumor progression, but melatonin weakened the tumor-promoting effect of CAFs. Compared with tumor cells, IL-8 was mainly expressed in CAFs. CAFs-overexpressing IL-8 induced the epithelial-mesenchymal transition (EMT) of tumor cells, and a positive crosstalk was observed between CAFs and tumor cells undergoing EMT, thereby further promoting the IL-8 expression. Melatonin suppressed this crosstalk by inhibiting the NF-κB pathway, thereby impeding the IL-8 expression from CAFs. Importantly, melatonin reversed CAFs-derived IL-8-mediated EMT by inhibiting the AKT pathway. Melatonin was found to directly and indirectly inhibit tumor progression. CONCLUSION: Our research reveals the potential action mechanism of melatonin in regulating the CAF-tumor cell interaction and suggests the potential of melatonin as an adjuvant of tumor therapy.

Calcipotriol abrogates cancer-associated fibroblast-derived IL-8-mediated oxaliplatin resistance in gastric cancer cells via blocking PI3K/Akt signaling.

Activation of vitamin D receptor (VDR) in cancer-associated fibroblasts (CAFs) has been implicated in hesitating tumor progression and chemoresistance of several human malignancies. Yet, the role of VDR in CAF-induced chemotherapy resistance of gastric cancer (GC) cells remains elusive. In this study we first conducted immunohistochemistry analysis on tissue microarrays including 88 pairs of GC and normal mucosa samples, and provided clinical evidence that VDR was mainly expressed in gastric mucous cells but almost invisible in CAFs, and VDR expression was negatively correlated with malignant clinical phenotype and advanced stages, low VDR expression confers to poor overall survival rate of patients with GC. In a co-culture system of primary CAFs and cancer cells, we showed that treatment of HGC-27 and AGS GC cells with VDR ligand calcipotriol (Cal, 500 nM) significantly inhibited CAF-induced oxaliplatin resistance. By using RNA-sequencing and Human Cytokine Antibody Array, we demonstrated that IL-8 secretion from CAFs induced oxaliplatin resistance via activating the PI3K/AKT pathway in GC, whereas Cal treatment greatly attenuated the tumor-supportive effect of CAF-derived IL-8 on GC cells. Taken together, this study verifies the specific localization of VDR in GC tissues and demonstrates that activation of VDR abrogates CAF-derived IL-8-mediated oxaliplatin resistance in GC via blocking PI3K/Akt signaling, suggesting vitamin D supplementation as a potential strategy of enhancing the anti-tumor effect of chemotherapy in GC.

Inclusion of cancer-associated fibroblasts in drug screening assays to evaluate pancreatic cancer resistance to therapeutic drugs.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by a pro-inflammatory stroma and multi-faceted microenvironment that promotes and maintains tumorigenesis. However, the models used to test new and emerging therapies for PDAC have not increased in complexity to keep pace with our understanding of the human disease. Promising therapies that pass pre-clinical testing often fail in pancreatic cancer clinical trials. The objective of this study was to investigate whether changes in the drug-dosing regimen or the addition of cancer-associated fibroblasts (CAFs) to current existing models can impact the efficacy of chemotherapy drugs used in the clinic. Here, we reveal that gemcitabine and paclitaxel markedly reduce the viability of pancreatic cell lines, but not CAFs, when cultured in 2D. Following the use of an in vitro drug pulsing experiment, PDAC cell lines showed sensitivity to gemcitabine and paclitaxel. However, CAFs were less sensitive to pulsing with gemcitabine compared to their response to paclitaxel. We also identify that a 3D co-culture model of MIA PaCa-2 or PANC-1 with CAFs showed an increased chemoresistance to gemcitabine when compared to standard 2D mono-cultures a difference to paclitaxel which showed no measurable difference between the 2D and 3D models, suggesting a complex interaction between the drug in study and the cell type used. Changes to standard 2D mono-culture-based assays and implementation of 3D co-culture assays lend complexity to established models and could provide tools for identifying therapies that will match clinically the success observed with in vitro models, thereby aiding in the discovery of novel therapies.

Biejiajian pill inhibits progression of hepatocellular carcinoma by downregulating PDGFRß signaling in cancer-associated fibroblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Biejiajian pill (BJJP) is a canonical formula that is clinically used to treat chronic liver disease, especially to decrease the incidence of hepatocellular carcinoma (HCC). However, the mechanisms underlying the prevention of HCC progression by BJJP remain unclear. AIM OF THE STUDY: This study aimed to determine whether BJJP inhibits HCC progression by downregulating platelet-derived growth factor receptor beta (PDGFRß) signaling in cancer-associated fibroblasts (CAFs) in a mouse model of diethylnitrosamine (DEN)/carbon tetrachloride (CCl4)-induced HCC. MATERIALS AND METHODS: C57BL/6 male mice were intraperitoneally injected with DEN 2 weeks after birth, followed by repeated injections of CCl4 weekly from 6 weeks of age onwards, to recapitulate features of HCC. At week 14, BJJP was orally administered to mice. The effects of BJJP on HCC progression were evaluated using histology, immunohistochemistry, and serum biochemical marker levels. Transcriptome analysis, molecular docking, quantitative real-time PCR, and Western blot were used to study the genes targeted by BJJP and the associated signaling pathway. The effects of BJJP on PDGFRß signaling in CAFs and the underlying mechanism were demonstrated. RESULTS: BJJP treatment significantly suppressed carcinogenesis and cancer progression, and it ameliorated liver inflammation in mice with HCC. A total of 176 genes, including PDGFRß, were significantly downregulated after BJJP treatment and five components of BJJP with high binding affinity to PDGFRß were identified. BJJP inhibited the phosphorylation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and glycogen synthase kinase 3 beta (GSK3ß) by suppressing PDGFRß expression in CAFs, and it also downregulated the expression of the downstream proteins hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGF-A). Furthermore, BJJP-containing serum consistently reduced PDGFRß, HGF, and VEGF-A expression levels in HSC-derived CAFs in vitro. Importantly, PDGF-BB induced PDGFRß activation in CAFs and both BJJP and sunitinib (a kinase inhibitor) inhibited PDGF-BB/PDGFRß signaling. CONCLUSION: BJJP inhibits the progression of HCC through suppressing VEGF-A and HGF expression in CAFs by downregulating PDGFRß signaling.

Near-Infrared Photoimmunotherapy Targeting Podoplanin-Expressing Cancer Cells and Cancer-Associated Fibroblasts.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that uses an antibody-IRDye700DX (IR700) conjugate that binds to a target followed by the application of NIR light that results in dramatic changes in solubility of the conjugate leading to rapid cell membrane damage and highly immunogenic cell death. NIR-PIT has been used clinically in treating advanced head and neck cancers using an anti-EGFR antibody-IR700 conjugate and has been conditionally approved for clinical use in Japan. NIR-PIT can be employed using a wide range of targeting antibodies. Podoplanin (PDPN), also known as gp38, is a 38 kDa type-1 transmembrane protein associated with lymphatic vessels. In cancer cells and cancer-associated fibroblasts (CAFs), PDPN expression has been widely reported and correlates with poor outcomes in several cancer types. In this study, we evaluated the efficacy of PDPN-targeted NIR-PIT in syngenetic mouse models of cancer. PDPN-targeted NIR-PIT destroyed PDPN-expressing cancer cells and CAFs selectively, suppressing tumor progression and prolonging survival with minimal damage to lymphatic vessels compared with the control group. Interestingly, PDPN-targeted NIR-PIT also exerted a therapeutic effect by targeting CAFs in tumor models which do not express in cancer cells. Furthermore, increased cytotoxic T cells in the tumor bed after PDPN-targeted NIR-PIT were observed, suggesting enhanced host antitumor immunity. Thus, PDPN-targeted NIR-PIT is a promising new cancer therapy strategy for PDPN-expressing cancer cells and CAFs.

Dual-targeted near-infrared photoimmunotherapy for esophageal cancer and cancer-associated fibroblasts in the tumor microenvironment.

Cancer-associated fibroblasts (CAFs) play a significant role in tumor progression within the tumor microenvironment. Previously, we used near-infrared photoimmunotherapy (NIR-PIT), a next-generation cancer cell-targeted phototherapy, to establish CAF-targeted NIR-PIT. In this study, we investigated whether dual-targeted NIR-PIT, targeting cancer cells and CAFs, could be a therapeutic strategy. A total of 132 cases of esophageal cancer were analyzed for epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), and fibroblast activation protein (FAP) expression using immunohistochemistry. Human esophageal cancer cells and CAFs were co-cultured and treated with single- or dual-targeted NIR-PIT in vitro. These cells were co-inoculated into BALB/c-nu/nu mice and the tumors were treated with single-targeted NIR-PIT or dual-targeted NIR-PIT in vivo. Survival analysis showed FAP- or EGFR-high patients had worse survival than patients with low expression of FAP or EGFR (log-rank, P < 0.001 and P = 0.074, respectively), while no difference was observed in HER2 status. In vitro, dual (EGFR/FAP)-targeted NIR-PIT induced specific therapeutic effects in cancer cells and CAFs along with suppressing tumor growth in vivo, whereas single-targeted NIR-PIT did not show any significance. Moreover, these experiments demonstrated that dual-targeted NIR-PIT could treat cancer cells and CAFs simultaneously with a single NIR light irradiation. We demonstrated the relationship between EGFR/FAP expression and prognosis of patients with esophageal cancer and the stronger therapeutic effect of dual-targeted NIR-PIT than single-targeted NIR-PIT in experimental models. Thus, dual-targeted NIR-PIT might be a promising therapeutic strategy for cancer treatment.

MEG3 Expression Indicates Lymph Node Metastasis and Presence of Cancer-Associated Fibroblasts in Papillary Thyroid Cancer.

Papillary thyroid cancer is the most common endocrine malignancy, occurring at an incidence rate of 12.9 per 100,000 in the US adult population. While the overall 10-year survival of PTC nears 95%, the presence of lymph node metastasis (LNM) or capsular invasion indicates the need for extensive neck dissection with possible adjuvant radioactive iodine therapy. While imaging modalities such as ultrasound and CT are currently in use for the detection of suspicious cervical lymph nodes, their sensitivities for tumor-positive nodes are low. Therefore, advancements in preoperative detection of LNM may optimize the surgical and medical management of patients with thyroid cancer. To this end, we analyzed bulk RNA-sequencing datasets to identify candidate markers highly predictive of LNM. We identified MEG3, a long-noncoding RNA previously described as a tumor suppressor when expressed in malignant cells, as highly associated with LNM tissue. Furthermore, the expression of MEG3 was highly predictive of tumor infiltration with cancer-associated fibroblasts, and single-cell RNA-sequencing data revealed the expression of MEG3 was isolated to cancer-associated fibroblasts (CAFs) in the most aggressive form of thyroid cancers. Our findings suggest that MEG3 expression, specifically in CAFs, is highly associated with LNM and may be a driver of aggressive disease.

Jianpi Jiedu Recipe inhibits colorectal cancer liver metastasis via regulating ITGBL1-rich extracellular vesicles mediated activation of cancer-associated fibroblasts.

BACKGROUND: Extracellular vesicles (EVs) contribute greatly to the formation of pre-metastatic niche and tumor metastasis. Our previous study has revealed that tumor-derived ITGBL1 (integrin beta- like 1)-rich EVs activate fibroblasts through the NF-κB signaling to promote colorectal cancer (CRC) metastasis. Targeting ITGBL1-loaded EVs may be a new and effective therapy for treating CRC metastasis. Simultaneously, our preliminary clinical trial has demonstrated that Jianpi Jiedu Recipe (JPJDR) was an ideal alternative traditional Chinese medicine for the prevention and treatment of CRC metastasis. However, the underlying mechanism of JPJDR in the prevention of CRC metastasis is not clear. In this study, we will investigate the regulatory effect of JPJDR on ITGBL1 levels in CRC-derived EVs, and to detect how JPJDR regulate ITGBL1-rich EVs mediated activation of fibroblasts to inhibit CRC metastasis. METHODS: EVs derived from CRC cells with/without JPJDR treatment were obtained by ultracentrifugation, following by characterization with electron microscopy, LM10 nanoparticle characterization system and western blot. The migration and growth of CRC cells were tested by transwell assay, wound healing assay and colony formation assay. The effect of JPJDR on the fibroblasts-activation associated inflammatory factors including IL-6, IL-8 and α-SMA was detected by real-time PCR. The levels of IL-6, IL-8 and α-SMA in the cell culture supernatant were detected by ELISA. The protein expressions of TNFAIP3, ITGBL1, p-NF-κB, IκBα and ß-actin were detected by western blot. Liver metastasis model in mice was established by injecting MC38 single cell suspension into the spleen of mice to observe the effect of JPJDR on CRC liver metastasis. Immunohistochemistry were applied to detect the expression of ITGBL1 and TNFAIP3 in the liver metastatic tissues. Tissue immunofluorescence detection was performed to observe the regulatory effect of JPJDR on the ITGBL1-NF-κB signaling pathway. Cancer-associated fibroblasts (CAFs) in the liver metastatic tissues were sorted and characterized by platelet-derived growth factor receptor ß (PDGFRß) with flow cytometry, following by the detection of inflammatory factors including IL-6, IL-8 and α-SMA using real-time PCR. RESULTS: JPJDR reduced the ITGBL1 levels in CRC cells-derived EVs. JPJDR inhibited the migration and growth of CRC cells via regulating ITGBL1-rich EVs mediated fibroblasts activity. Mechanically, JPJDR decreased fibroblasts activation by regulating ITGBL1-rich EVs mediated TNFAIP3-NF-κB signaling. Further in vivo experiments demonstrated that JPJDR reduced CRC liver metastasis by regulating ITGBL1-rich EVs secretion from CRC and blocked the fibroblasts activation by regulating ITGBL1-TNFAIP3- NF-κB signaling. CONCLUSION: Our research demonstrated that JPJDR preventd CRC liver metastasis via down-regulating CRC-derived ITGBL1-loaded EVs mediated activation of CAFs, providing the experimental evidence for the clinical application of JPJDR in the prevention and treatment of CRC metastasis. More importantly, our study confirmed the great benefits of therapeutic targeting the EVs-mediated metastasis and warranted future clinical validation.

Targeting the tumor microenvironment of pancreatic ductal adenocarcinoma using nano-phytomedicines.

Despite advanced therapeutic strategies, the mortality and morbidity of pancreatic cancer (PC) have been increasing. This is due to the anomalous proliferation activity of stromal cells, like cancer-associated fibroblasts (CAFs), in the tumor microenvironment (TME). These cells develop resistance in the tumor cells, blocking the drug from entering the target tumor site, ultimately resulting in tumor metastasis. Additionally, the current conventional adjuvant techniques, including chemo and radiotherapy, carry higher risk due to their excess toxicity against normal healthy cells. Phytochemicals including curcumin, irinotecan and paclitaxel are anti-oxidants, less toxic, and have anti-cancerous properties; however, the use of phytochemicals is limited due to their less solubility and bioavailability. Nanotechnology offers the resources to directly target the drug to the tumor site, thereby enhancing the therapeutic efficacy of the current treatment modalities. This review focuses on the importance of nanotechnology for pancreatic ductal adenocarcinoma (PDAC) therapy and on delivering the nano-formulated phytochemicals to the target site.

Aesculus hippocastanum L. extract differently modulates normal human dermal fibroblasts and cancer-associated fibroblasts from basal/squamous cell carcinoma.

Fibroblasts are actively involved in the formation of granulation tissue and/or tumor stroma. These cells possess the potential to differentiate into myofibroblasts acquiring a highly contractile phenotype characterized by the expression of α-smooth muscle actin (SMA). Considering TGF-ß1 as the main inducer of myofibroblast differentiation and horse chestnut extract (HCE) as an effective modulator of the wound healing, we have new evidence to demonstrate canonical TGF-ß1/SMAD and non-canonical/non-SMAD signaling in normal fibroblasts, isolated from healthy human skin (human dermal fibroblasts - HDFs), and their malignant counterparts (CAFs) isolated from basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) using western blot and immunofluorescence. Our study revealed that HCE stimulated the deposition of fibronectin by BCC fibroblasts (BCCFs), an effect not seen in other studied fibroblasts. Moreover, HCE in combination with TGF-ß1 showed a synergic effect on the presence of polymerized SMA-stress fibers, particularly visible in CAFs. Interestingly, the TGF-ß1 exposure led to activation of the canonical SMAD signaling in HDFs and BCCFs, whereas treatment of SCC fibroblasts (SCCFs) resulted in activation of the non-canonical AKT and/or ERK1/2 signaling. In conclusion, we observed specific differences in signaling between HDFs and CAFs that should be considered when developing new therapeutic approaches targeting wound/tumor microenvironments.

Tumor microenvironment-based screening repurposes drugs targeting cancer stem cells and cancer-associated fibroblasts.

The tumorous niche may drive the plasticity of heterogeneity and cancer stemness, leading to drug resistance and metastasis, which is the main reason of treatment failure in most cancer patients. The aim of this study was to establish a tumor microenvironment (TME)-based screening to identify drugs that can specifically target cancer stem cells (CSCs) and cancer-associated fibroblasts (CAFs) in the TME. Methods: Lung cancer patient-derived cancer cell and CAFs were utilized to mimic the TME and reproduce the stemness properties of CSCs in vitro and develop a high-throughput drug screening platform with phenotypical parameters. Limiting dilution assay, sphere-forming and ALDH activity assay were utilized to measure the cancer stemness characteristics. In vivo patient-derived xenograft (PDX) models and single-cell RNA sequencing were used to evaluate the mechanisms of the compounds in CSCs and CAFs. Results: The TME-based drug screening platform could comprehensively evaluate the response of cancer cells, CSCs and CAFs to different treatments. Among the 1,524 compounds tested, several drugs were identified to have anti-CAFs, anticancer and anti-CSCs activities. Aloe-emodin and digoxin both show anticancer and anti-CSCs activity in vitro and in vivo, which was further confirmed in the lung cancer PDX model. The combination of digoxin and chemotherapy improved therapeutic efficacy. The single-cell transcriptomics analysis revealed that digoxin could suppress the CSCs subpopulation in CAFs-cocultured cancer cells and cytokine production in CAFs. Conclusions: The TME-based drug screening platform provides a tool to identify and repurpose compounds targeting cancer cells, CSCs and CAFs, which may accelerate drug development and therapeutic application for lung cancer patients.

The functional cross talk between cancer cells and cancer associated fibroblasts from a cancer mechanics perspective.

The function of biological tissues in health and disease is regulated at cellular level and is highly influenced by the physical microenvironment, through the interaction of forces between cells and ECM, which are perceived through mechanosensing pathways. In cancer, both chemical and physical signaling cascades and their interactions are involved during cell-cell and cell-ECM communications to meet requirements of tumor growth. Among stroma cells, cancer associated fibroblasts (CAFs) play key role in tumor growth and pave the way for cancer cells to initiate metastasis and invasion to other tissues, and without recruitment of CAFs, the process of cancer invasion is dysfunctional. This is through an intense chemical and physical cross talks with tumor cells, and interactive remodeling of ECM. During such interaction CAFs apply traction forces and depending on the mechanical properties, deform ECM and in return receive physical signals from the micromechanical environment. Such interaction leads to ECM remodeling by manipulating ECM structure and its mechanical properties. The results are in form of deposition of extra fibers, stiffening, rearrangement and reorganization of fibrous structure, and degradation which are due to a complex secretion and expression of different markers triggered by mechanosensing of tumor cells, specially CAFs. Such events define cancer progress and invasion of cancer cells. A systemic knowledge of chemical and physical factors provides a holistic view of how cancer process and enhances the current treatment methods to provide more diversity among targets that involves tumor cells and ECM structure.

Active Tumoral/Tumor Environmental Dual-Targeting by Non-Covalently Arming with Trispecific Antibodies or Dual-Bispecific Antibodies on Docetaxel-Loaded mPEGylated Nanocarriers to Enhance Chemotherapeutic Efficacy and Minimize Systemic Toxicity.

PURPOSE: This study was aimed at developing the trispecific antibodies (anti-EGFR/anti-FAP/anti-mPEG, TsAb) or dual bispecific antibodies (anti-EGFR/anti-mPEG and anti-FAP/anti-mPEG) docetaxel (DTX)-loaded mPEGylated lecithin-stabilized micelles (mPEG-lsbPMs) for improving the targeting efficiency and therapeutic efficacy. METHODS: mPEG-lsbPMs were simply prepared via thin film method. The trispecific antibodies or bispecific antibodies bound the mPEG-lsbPMs by anti-mPEG Fab fragment. The formulations were characterized by DLS and TEM; in vitro and in vivo studies were also conducted to evaluate the cellular uptake, cell cytotoxicity and therapeutic efficacy. RESULTS: The particle sizes of mPEG-lsbPMs with or without the antibodies were around 100 nm; the formulations showed high encapsulation efficiencies of 97.12%. The TsAb and dual bispecific antibodies were fabricated and demonstrated their targeting ability. Two EGFR-overexpressed cell lines (HT-29 and MIA PaCa-2) were co-cultured with FAP-overexpressed WS1 cells (HT-29/WS1; MIA PaCa-2/WS1) to mimic a tumor coexisting in the tumor microenvironment. Cellular binding study revealed that the binding of anti-FAP micelles to three co-culture ratios (4:1, 1:1, and 1:4) of HT-29/EGFR to WS1/FAP was significantly higher than that for TsAb micelles and dual (1:1) micelles, and the binding of those targeting antibodies to WS1/FAP and MIA PaCa-2/EGFR was equally efficacious resulting in a similar binding amount of the TsAb and dual BsAbs (1:1) with the co-culture of MIA PaCa-2/EGFR and WS1/FAP at a 1:1 ratio. Antitumor efficacy study showed that treatment with DTX-loaded mPEG-lsbPMs modified with or without BsAbs, dual BsAbs (1:1), and TsAbs was enhanced in inhibiting tumor growth compared with that for Tynen® while showing fewer signs of adverse effects. CONCLUSION: Active targeting of both tumors and TAF-specific antigens was able to increase the affinity of DTX-loaded mPEG-lsbPMs toward tumor cells and TAFs leading to successive uptake by tumor cells or TAFs which enhanced their chemotherapeutic efficacy against antigen-positive cancer cells.