Nursing practice to fulfill the information needs of parents of hospitalized children with cancer and related factors.

PURPOSE: This study aimed to clarify the current status of nursing practice to fulfill the information needs of parents of hospitalized children with cancer and to determine the associated factors. DESIGN AND METHODS: A cross-sectional survey using a questionnaire was administered to nurses working on wards admitting children with cancer in Japan. Data were analyzed using logistic regression analysis, after exploratory factor analysis. RESULTS: Three factors were extracted as nursing practice: "provision of information that supports the child's future and other family members' daily lives" (factor 1), "provision of information regarding care for the child in the treatment process" (factor 2), "provision of information regarding the child's disease and treatment" (factor 3). Among these three factors, factor 1 achieved the lowest score for the level of practice. Logistic regression analysis indicated that interprofessional information sharing increased the scores of factors 1 and 3 (Odds ratio: 6.150, and 4.932, respectively); assessment of parental information needs increased the scores of factors 1, 2, and 3 (Odds ratio: 3.993, 3.654, and 3.671, respectively); and participation in training increased the score of factor 2 (Odds ratio: 3.078). CONCLUSIONS: Nursing practice to fulfill the parents' information needs consisted of three factors. The degree of practice varied according to the information content and was primarily influenced by assessment of parental information needs, interprofessional information sharing, and participation in training. PRACTICE IMPLICATIONS: It is necessary for nurses to accurately assess parents' needs, and interprofessional sharing of information is important to fulfill the information needs of parents.

Influence of Intestinal Barrier on Alleviating an Increase in Blood Pressure by Sodium Alginate Intake in 2-Kidney, 1-Clip Renovascular Hypertensive Rats.

Sodium alginate (SALG) is a substance derived from brown seaweed that has been shown to reduce blood pressure (BP). However, its effects on renovascular hypertension caused by 2-kidney, 1-clip (2K1C) are not yet clear. Previous research suggests that hypertensive rats have increased intestinal permeability, and that SALG improves the gut barrier in inflammatory bowel disease mouse models. Therefore, the goal of this study was to determine whether the antihypertensive effects of SALG involve the intestinal barrier in 2K1C rats. Rats were fed either a 1.0% SALG diet or a control diet for six weeks after being subjected to 2K1C surgery or a sham operation. The systolic BP was measured weekly, and the mean arterial BP was measured at the end of the study. Intestinal samples were taken for analysis, and plasma lipopolysaccharide (LPS) levels were measured. The results showed that BP in 2K1C rats was significantly higher than in SHAM rats when fed CTL, but not when fed SALG. The gut barrier in 2K1C rats was improved by SALG intake. Plasma LPS levels also differed depending on the animal model and diet. In conclusion, dietary SALG may alleviate 2K1C renovascular hypertension by altering the gut barrier.

Prognostic impact and gene expression analysis of peri-tumoral alveolar macrophage in resected lung adenocarcinoma.

The prognostic significance and role of extratumoral alveolar macrophages (exAMs) in lung adenocarcinoma (LUAD) patients remain unknown. In this study, we investigated the prognostic impact and gene expression of exAMs in LUAD patients. The density of alveolar macrophages (AMs) in the peri-tumoral lung field (p-exAMs) and distant lung field (d-exAMs) was evaluated in 217 LUAD patients with lymph node metastasis. Patients with high p-exAMs showed significantly shorter recurrence-free (RFS) and shorter overall survival (OS) than those with low p-exAMs (p = 0.02 and p = 0.03, respectively), whereas there was no survival difference between patients with high d-exAMs and those with low d-exAMs. Multivariate analysis revealed that high p-exAMs was an independent predictive factor for RFS (HR: 1.54; 95% confidence interval [CI]:1.10-2.16; p = 0.01). Later, we collected AMs from the tumor periphery and distant segments in 13 resected lungs by bronchoalveolar lavage (BAL) procedure and compared mRNA expression. AMs in the tumor periphery expressed significantly higher levels of IL-10 and CCL2 than those in the distant segment (p < 0.01 and p = 0.03, respectively). Additionally, IL-10 and CCL2 significantly induced the growth and migration of the PC9 cells in vitro. This study suggests that p-exAMs should be considered as a tumor-promoting component in the tumor microenvironment.

Clinicopathological significance of peritumoral alveolar macrophages in patients with resected early-stage lung squamous cell carcinoma.

INTRODUCTION: This study aimed to clarify the correlation between the number of AMs and prognosis and to examine the gene expression of AMs in lung squamous cell carcinoma (SqCC). METHODS: We reviewed 124 stage I lung SqCC cases in our hospital and 139 stage I lung SqCC cases in The Cancer Genome Atlas (TCGA) cohort in this study. We counted the number of AMs in the peritumoral lung field (P-AMs) and in the lung field distant from the tumor (D-AMs). Moreover, we performed a novel ex vivo bronchoalveolar lavage fluid (BALF) analysis to select AMs from surgically resected lung SqCC cases and examined the expression of IL10, CCL2, IL6, TGFß, and TNFα (n = 3). RESULTS: Patients with high P-AMs had significantly shorter overall survival (OS) (p < 0.01); however, patients with high D-AMs did not have significantly shorter OS. Moreover, in TCGA cohort, patients with high P-AMs had a significantly shorter OS (p < 0.01). In multivariate analysis, a higher number of P-AMs were an independent poor prognostic factor (p = 0.02). Ex vivo BALF analysis revealed that AMs collected from the tumor vicinity showed higher expression of IL10 and CCL2 than AMs from distant lung fields in all 3 cases (IL-10: 2.2-, 3.0-, and 10.0-fold; CCL-2: 3.0-, 3.1-, and 3.2-fold). Moreover, the addition of recombinant CCL2 significantly increased the proliferation of RERF-LC-AI, a lung SqCC cell line. CONCLUSION: The current results indicated the prognostic impact of the number of peritumoral AMs and suggested the importance of the peritumoral tumor microenvironment in lung SqCC progression.

Cancer-associated fibroblast-dependent and -independent invasion of gastric cancer cells.

PURPOSE: Cancer cells are known to exhibit a cancer-associated fibroblast (CAF)-dependent invasive mode in the presence of CAFs. The purpose of this study was to investigate whether intrinsic factors of gastric cancer cells influence the CAF-dependent invasive mode of cancer cells. METHODS: We observed dynamic movement of CAFs, and cancer cells, by time-lapse imaging of 2-D and 3-D collagen invasion models, and evaluated invasion modes of gastric cancer cell lines (MKN-7, MKN-45, and HSC44PE). We further examined whether modification of invasive capacity of CAFs can alter invasive mode of MKN-7, and HSC44PE cells. RESULTS: When MKN-7 and MKN-45 cells were co-cultured with CAFs, CAFs first invade collagen matrix followed by cancer cells (CAF-dependent invasion), whereas HSC44PE cells invaded collagen matrix independent of CAFs' invasion. Overexpression or suppression of podoplanin in CAFs, respectively, increased or decreased the invasive capacity of CAFs, and significantly increased or decreased the number of invading MKN-7 cells, respectively. CAFs overexpressing a podoplanin mutant, lacking the cytoplasmic domain, had significantly reduced invasive capacity, compared to CAFs overexpressing wild-type podoplanin, and it also reduced the number of invading MKN-7 cells significantly. When HSC44PE cells, and CAFs were co-cultured, changes in the podoplanin expression in CAFs similarly altered the invasive capacity of CAFs, but it did not affect the number of invading HSC44PE cells. CONCLUSIONS: These results indicate that in presence of CAFs, gastric cancer cells exhibit both CAF-dependent and -independent modes of invasion, the determinants of which may depend on the intrinsic properties of the gastric cancer cells.

Polymeric micelles effectively reprogram the tumor microenvironment to potentiate nano-immunotherapy in mouse breast cancer models.

Nano-immunotherapy improves breast cancer outcomes but not all patients respond and none are cured. To improve efficacy, research focuses on drugs that reprogram cancer-associated fibroblasts (CAFs) to improve therapeutic delivery and immunostimulation. These drugs, however, have a narrow therapeutic window and cause adverse effects. Developing strategies that increase CAF-reprogramming while limiting adverse effects is urgent. Here, taking advantage of the CAF-reprogramming capabilities of tranilast, we developed tranilast-loaded micelles. Strikingly, a 100-fold reduced dose of tranilast-micelles induces superior reprogramming compared to free drug owing to enhanced intratumoral accumulation and cancer-associated fibroblast uptake. Combination of tranilast-micelles and epirubicin-micelles or Doxil with immunotherapy increases T-cell infiltration, resulting in cures and immunological memory in mice bearing immunotherapy-resistant breast cancer. Furthermore, shear wave elastography (SWE) is able to monitor reduced tumor stiffness caused by tranilast-micelles and predict response to nano-immunotherapy. Micellar encapsulation is a promising strategy for TME-reprogramming and SWE is a potential biomarker of response.

Drug-exposed cancer-associated fibroblasts facilitate gastric cancer cell progression following chemotherapy.

BACKGROUND: Cancer progression following chemotherapy is a significant barrier to effective cancer treatment. We aimed to evaluate the role of drug-exposed cancer-associated fibroblasts (CAFs) in the growth and progression of drug-exposed gastric cancer (GC) cells and to explore the underlying molecular mechanism. METHODS: The human GC cell line 44As3 and CAFs were treated with 5-fluorouracil and oxaliplatin (5FU + OX). 5FU + OX-pretreated 44As3 cells were then cultured in a conditioned medium (CM) from 5FU + OX-pretreated CAFs, and the growth and migration/invasion ability of the cells were evaluated. We also compared the clinicopathological characteristics of the GC patients treated with S1 + OX in accordance with the properties of their resected specimens, focusing on the number of CAFs. Changes in gene expression in CAFs and 44As3 cells were comprehensively analyzed using RNA-seq analysis. RESULTS: The CM from 5FU + OX-pretreated CAFs promoted the migration and invasion of 5FU + OX-pretreated 44As3 cells. Although the number of cases was relatively small (n = 21), the frequency of positive cases of lymphovascular invasion and the recurrence rate were significantly higher in those with more residual CAF. RNA-seq analysis revealed 5FU + OX-pretreated CAF-derived glycoprotein 130 (gp130) as a candidate factor contributing to the increased migration of 5FU + OX-pretreated 44As3 cells. Administration of the gp130 inhibitor SC144 prevented the increased migration ability of 5FU + OX-pretreated 44As3 cells owing to drug-treated CAFs. CONCLUSIONS: Our findings provide evidence regarding the interactions between GC cells and CAFs in the tumor microenvironment following chemotherapy, suggesting that ligands for gp130 may be novel therapeutic targets for suppressing or preventing metastasis in GC.

Relationship between podoplanin-expressing cancer-associated fibroblasts and the immune microenvironment of early lung squamous cell carcinoma.

AIM: Cancer-associated fibroblasts (CAFs) expressing podoplanin (PDPN) harbor a fibrous tumor microenvironment that promotes cancer progression in lung adenocarcinoma. In this study, we investigated whether tumor-promoting PDPN+ CAFs contribute to the immunosuppressive microenvironment in lung squamous cell carcinoma (SqCC). M&M: The gene expression profiles of immunosuppressive cytokines were compared using The Cancer Genome Atlas (TCGA) microarray lung SqCC data (n = 484) between a PDPN-high group and a PDPN-low group. Further, using patient-derived CAFs from surgically resected lung SqCC, the PDPN+ fraction was sorted and gene and protein expressions were analyzed. Finally, immunohistochemical staining was conducted on 131 surgically resected lung SqCC; CD8+ and FOXP3+ tumor infiltrating lymphocytes (TILs), and CD204+ tumor-associated macrophages (TAMs) were evaluated in cases with PDPN+ and PDPN- CAFs. RESULTS: Analysis of TCGA database revealed that the PDPN-high group exhibited significantly higher expression of interleukin (IL)-1A, IL-1B, IL-6, IL-10, monocyte chemoattractant protein-1 (CCL2), colony stimulating factor 1 (CSF1), fibroblast growth factor 2 (FGF2), galectin 1 (LGALS1), platelet derived growth factor subunit A (PDGFA), PDGFB, and transforming growth factor-ß1 (TGFB1) than those in the PDPN-low group. Among them, it was found that TGFB1 expression was higher in patient-derived PDPN+ CAFs. Immunohistochemical analyses revealed that more CD204+ TAMs infiltrated the tumor tissues in cases with PDPN+ CAFs than in cases with PDPN- CAFs (P <  0.03), while CD8+ and FOXP3+ TILs did not. Furthermore, in the same tumor, CD204+ TAMs infiltrated more in PDPN+ CAF-rich areas (P =  0.005). CONCLUSION: PDPN+ CAFs showed higher expression of TGFB1 and were associated with CD204+ TAM infiltration in stage-I lung SqCC, suggesting that PDPN+ CAFs were associated with the immunosuppressive tumor microenvironment.

Fibroblasts-dependent invasion of podoplanin-positive cancer stem cells in squamous cell carcinoma.

To clear whether podoplanin-positive cancer stem cells in squamous cell carcinoma have higher invasion activity during a fibroblasts-dependent invasion. A collagen gel invasion assay was performed using fluorescent ubiquitination-based cell cycle indicator-labeled A431 cells. The total number and number of invading cells in S/G2/M phase were counted using time-lapse imaging cocultured with fibroblasts. There was no significant difference between the number of invading podoplanin-positive and negative A431 cells when fibroblasts did not exist. On the contrary, the number of invading podoplanin-positive cells was significantly higher when fibroblasts existed. The frequency of cells in S/G2/M phase among invasion was no difference. Knockdown of podoplanin decreased the number of invaded A431 cells significantly when fibroblasts existed. Podoplanin-positive A431 cells display higher invasion activity when fibroblasts exist, suggesting that some biological functions of cancer stem cells might become evident only within the fibrous tumor microenvironment.

Renal TNFα activates the WNK phosphorylation cascade and contributes to salt-sensitive hypertension in chronic kidney disease.

The inappropriate over-activation of the with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-sodium chloride cotransporter (NCC) phosphorylation cascade increases sodium reabsorption in distal kidney nephrons, resulting in salt-sensitive hypertension. Although chronic kidney disease (CKD) is a common cause of salt-sensitive hypertension, the involvement of the WNK phosphorylation cascade is unknown. Moreover, the effect of immune systems on WNK kinases has not been investigated despite the fact that immune systems are important for salt sensitivity. Here we demonstrate that the protein abundance of WNK1, but not of WNK4, was increased at the distal convoluted tubules in the aristolochic acid nephropathy mouse model of CKD. Accordingly, the phosphorylation of both SPAK and NCC was also increased. Moreover, a high-salt diet did not adequately suppress activation of the WNK1-SPAK-NCC phosphorylation cascade in this model, leading to salt-sensitive hypertension. WNK1 also was increased in adenine nephropathy, but not in subtotal nephrectomy, models of CKD. By comparing the transcripts of these three models focusing on immune systems, we hypothesized that tumor necrosis factor (TNF)-α regulates WNK1 protein expression. In fact, TNF-α increased WNK1 protein expression in cultured renal tubular cells by reducing the transcription and protein levels of NEDD4-2 E3-ligase, which degrades WNK1 protein. Furthermore, the TNF-α inhibitor etanercept reversed the reduction of NEDD4-2 expression and upregulation of the WNK1-SPAK-NCC phosphorylation cascade in distal convoluted tubules in vivo in the aristolochic acid nephropathy model. Thus, salt-sensitive hypertension is induced in CKD via activation of the renal WNK1- SPAK-NCC phosphorylation cascade by TNF-α, reflecting a link with the immune system.

Dietary capsaicin-mediated attenuation of hypertension in a rat model of renovascular hypertension.

Background: Capsaicin, a pungent component of chili pepper, has been reported to decrease blood pressure (BP) and to cause vasorelaxation via nitric oxide (NO) production. However, it is still unclear how dietary capsaicin effects on renovascular hypertension. To examine this, we observed the effects of dietary capsaicin on BP in 2-kidney, 1-clip renovascular hypertension (2K1C) rats, and investigated the participation of NO in the mechanism.Methods: Rats with 2K1C or sham-operated rats (SHAM) were treated with 0.006% capsaicin diet (CAP) or control diet (CTL) for 6 weeks. Systolic BP (SBP) was measured by tail-cuff method once a week. In the end, mean arterial BP (MAP) was measured in the rats under anesthesia. These observations were performed also in the rats taking a NO synthase (NOS) inhibitor (LN). After rats were euthanized, thoracic aortas were collected and used for western blot analyses to evaluate the phosphorylated ratio of endothelial NOS (eNOS), protein kinase A (PKA) and B (Akt), in order to explore a mechanism of the effects on BP by dietary capsaicin.Results: SBP and MAP in 2K1C rats were significantly higher than in SHAM rats when fed CTL, but not when fed CAP. Those in 2K1C-CAP rats were significantly lower than in 2K1C-CTL rats. LN suppressed the effect of dietary capsaicin. The ratios of phosphorylated (p-) eNOS/eNOS and p-Akt/Akt, but not p-PKA/PKA, were significantly increased in rats fed CAP compared with rats fed CTL.Conclusion: Dietary capsaicin may alleviate 2K1C renovascular hypertension, probably via enhancing phosphorylation of Akt and eNOS.Abbreviations: 2K1C: 2-kidney, 1-clip hypertension model; Akt: protein kinase B; Ang II: angiotensin II; ANOVA: measures analysis of variance; BP: blood pressure; EC: endothelial cell; eNOS: endothelial nitric oxide synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L-NAME, LN: Nω-Nitro-L-arginine methyl ester hydrochloride; MA: mesenteric arteries; MAP: mean arterial blood pressure; NO: nitric oxide; PKA: protein kinase A; PVDF: polyvinylidene difluoride; SBP: Systolic blood pressure; SHR: spontaneously hypertensive rats; SN: sympathetic nervous; TRPV1: transient receptor potential vanilloid type 1; WKY: Wistar Kyoto rats.

Interaction between cancer cells and cancer-associated fibroblasts after cisplatin treatment promotes cancer cell regrowth.

Regrowth of cancer cells following chemotherapy is a significant problem for cancer patients. This study examined whether cancer-associated fibroblasts (CAFs), a major component of a tumor microenvironment, promote cancer cell regrowth after chemotherapy. First, we treated human lung adenocarcinoma cell line A549 and CAFs from four patients with cisplatin. Cisplatin treatment inhibited the viable cell number of A549 cells and induced epithelial-mesenchymal transition. After cisplatin was removed, A549 cells continued to manifest the mesenchymal phenotype and proliferated 2.2-fold in 4 days (regrowth of A549 cells). Cisplatin treatment inhibited the viable cell number of CAFs from four patients also. The CM (derived from cisplatin-pretreated CAFs from two patients) significantly enhanced the regrowth of cisplatin-pretreated A549 cells, and the CM derived from cisplatin-naïve CAFs marginally enhanced A549 regrowth. By contrast, the CM derived from either cisplatin-pretreated CAFs or cisplatin-naïve CAFs failed to enhance the growth of cisplatin-naïve A549 cells. The CM derived from cisplatin-pretreated CAFs did not enhance the proliferation of A549 cells in which epithelial-mesenchymal transition was induced by TGFß-1. Our findings indicate the possibility that humoral factors from cisplatin-pretreated CAFs promote the regrowth of cisplatin-pretreated A549 cells. These results suggest that interactions between cancer cells and CAFs may significantly enhance cancer cell regrowth within the tumor microenvironment after cisplatin treatment.

Secretion of high amounts of hepatocyte growth factor is a characteristic feature of cancer-associated fibroblasts with EGFR-TKI resistance-promoting phenotype: A study of 18 cases of cancer-associated fibroblasts.

Humoral factors from cancer-associated fibroblasts (CAFs) reportedly affect epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance in cancer cells with EGFR mutations. The aim of this study was to identify the robust humoral factors secreted from CAFs that induce the primary resistance to EGFR-TKI. We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell line (PC-9) treated with condition media (CM) from 18 cases of CAFs and matched non-cancerous-tissue-associated fibroblasts (NCAFs). We measured the expression levels of hepatocyte growth factor (HGF), interleukin-6, fibroblast growth factor-2, insulin-like growth factor-1, and vascular endothelial growth factor-A in CAFs and NCAFs. We examined whether HGF neutralizing antibody could annul the EGFR-TKI resistance induced by CM from CAFs. Compared to CM from NCAFs, CM from CAFs increased the resistance of PC-9 cells to EGFR-TKI in five out of 18 cases. Relative expression ratio of HGF messenger RNA was significantly higher in these five CAFs compared to others (P = 0.0013), whereas other cytokines were not. In four of these five cases, the addition of HGF neutralizing antibody significantly decreased the survival ratio of PC-9 cells. This study suggests that the secretion of higher amounts of HGF is the robust feature of EGFR-TKI resistance-promoting CAFs.

Organoid culture containing cancer cells and stromal cells reveals that podoplanin-positive cancer-associated fibroblasts enhance proliferation of lung cancer cells.

OBJECTIVE: Podoplanin-positive cancer-associated fibroblasts (CAFs) play an important role in tumor progression. The aim of this study was to evaluate the effect of podoplanin (+) CAFs on the proliferation of cancer cells using a three-dimensional (3D) organoid model. MATERIALS AND METHODS: We examined the success rate of organoid culture containing PC-9 cancer cells and CAFs. Thereafter, we compared the proliferating index (MIB-1 index) of PC-9 cells co-cultured with podoplanin-overexpressing CAFs and control CAFs using organoid specimens. Furthermore, we compared the MIB-1 labeling index of cancer cells in podoplanin (+) CAFs cases (n = 13) and podoplanin (-) CAFs cases (n = 14) using surgically resected adenocarcinoma specimens. RESULTS: Without CAFs, PC-9 cells did not form any organoid (success rate: 0%). When PC-9 cells were mixed with CAFs (1:10), the mixed cells generated round and steric aggregates (hybrid cancer organoids, success rate: 100%). In three independent experiments, the MIB-1 index of PC-9 cells in hybrid cancer organoids containing podoplanin-overexpressing CAFs was significantly higher than that of PC-9 cells in organoids containing control CAFs (Exp. 1: 40.4% vs. 24.4%; Exp. 2: 40.0% vs. 24.5%; Exp. 3: 40.3% vs. 25.2%; p < 0.001). Surgically resected human tumors revealed that the MIB-1 index of adenocarcinoma cells was significantly higher in the case of podoplanin (+) CAFs than in the case of podoplanin (-) CAFs (34.8% vs. 16.2%; p < 0.01). CONCLUSION: Our data suggested that the hybrid cancer organoid model might reflect the growth-promoting effect of podoplanin (+) CAFs in cancer cells, and this new system can be a useful tool for evaluating the tumor microenvironment.

Spatiotemporal characteristics of fibroblasts-dependent cancer cell invasion.

PURPOSE: Cancer cells can invade the surrounding stroma with the aid of fibroblasts (fibroblasts-dependent invasion). The aim of this study was to explore the spatiotemporal characteristics of fibroblast-dependent invasion of cancer cells. METHODS: We performed an in vitro three-dimensional collagen invasion assay using Fluorescent Ubiquitination-based Cell Cycle indicator (Fucci)-labeled A431 carcinoma cells co-cultured with fibroblasts. We used time-lapse imaging to analyze the total cell number, frequencies of small cancer cell nests and S/G2/M phase of A431 cells in the invasion area. We compared the frequencies of small cancer cell nests and geminin (+) cancer cells within fibroblast-rich areas and fibroblast-poor areas in surgically resected human invasive squamous cell carcinoma tissue. RESULTS: The total invasion number of A431 cells was significantly higher when cultured with fibroblasts than without. The formation of small cancer cell nests was observed within the invasion area only in the presence of fibroblasts. The frequency of S/G2/M phase cells was significantly higher in A431 cells when cultured with fibroblasts than without. Immunohistochemical analysis of surgically resected human invasive squamous cell carcinoma tissue revealed that the frequencies of small cancer cell nests and geminin-positive cancer cells were significantly higher in fibroblast-rich areas compared to those in fibroblast-poor areas within the same tumor region. CONCLUSION: Our current study clearly showed that fibroblast-dependent cancer cell invasion was characterized by the progression in cell cycle and formation of small cancer cell nests.

Collagen type I induces EGFR-TKI resistance in EGFR-mutated cancer cells by mTOR activation through Akt-independent pathway.

Primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a serious problem in lung adenocarcinoma patients harboring EGFR mutations. The aim of this study was to examine whether and how collagen type I (Col I), the most abundantly deposited matrix in tumor stroma, affects EGFR-TKI sensitivity in EGFR-mutant cells. We evaluated the EGFR-TKI sensitivity of EGFR-mutated cancer cells cultured with Col I. Changes in the activation of downstream signaling molecules of EGFR were analyzed. We also examined the association between the Col I expression in tumor stroma in surgical specimens and EGFR-TKI response of postoperative recurrence patients with EGFR mutations. Compared to cancer cells without Col I, the survival rate of cancer cells cultured with Col I was significantly higher after EGFR-TKI treatment. In cancer cells cultured with and without Col I, EGFR-TKI suppressed the levels of phosphorylated (p-)EGFR, p-ERK1/2, and p-Akt. When compared to cancer cells without Col I, expression of p-P70S6K, a hallmark of mTOR activation, was dramatically upregulated in cancer cells with Col I. This activation was maintained even after EGFR-TKI treatment. Simultaneous treatment with EGFR-TKI and mTOR inhibitor abrogated Col I-induced resistance to EGFR-TKI. Patients with Col I-rich stroma had a significantly shorter progression-free survival time after EGFR-TKI therapy (238 days vs 404 days; P < .05). Collagen type I induces mTOR activation through an Akt-independent pathway, which results in EGFR-TKI resistance. Combination therapy using EGFR-TKI and mTOR inhibitor could be a possible strategy to combat this resistance.

A novel method to generate single-cell-derived cancer-associated fibroblast clones.

BACKGROUND: Cancer-associated fibroblasts (CAFs) communicate with cancer cells to play important roles in tumor progression. However, CAFs have heterogeneous phenotypes and functions. To understand how much of this heterogeneity relates to different biological responses, a more efficient method of generating single-cell-derived CAF clones is required. METHOD: We transduced two primary CAF cultures (CAFs-608 and CAFs-621) from lung adenocarcinoma with human telomerase reverse transcriptase (hTERT), mutant forms of cyclin dependent kinase 4 (CDK4R24C) independently and in combination (hTERT/CDK4R24C). After live imaging of each sorted-single cell, we evaluated the numbers of successfully established clones from CAFs-hTERT, CAFs-CDK4R24C, and CAFs-hTERT/CDK4R24C. Furthermore, we examined the expression levels of genes associated with tumor promoting pathways in established clones by qRT-PCR. RESULTS: Overexpression of hTERT and CDK4R24C efficiently extended the lifespan of both CAFs-608 and CAFs-621. The number of established CAF clones was highest for CAFs-hTERT/CDK4R24C, with 57 and 62 clones established from CAFs-608 and CAFs-621, respectively. Conversely, 16 and 11 CAFs-hTERT clones were derived from CAFs-608 and CAFs-621, respectively and 10 and 8 CAFs-CDK4R24C clones were from CAFs-608 and CAFs-621, respectively. TGF-b, ATCA2, and HSF1 mRNA levels differed in individual clones established from CAFs-hTERT/CDK4R24C. The expression levels of ATCA2 and HSF1 were much higher in one clone than in the other established clones and the parental CAFs. CONCLUSION: Our results show that combined exogenous expression of hTERT and mutant CDK4 is an effective method to generate single-cell-derived CAF clones. This provides an innovative and suitable approach to investigate the heterogeneous function and phenotype of CAFs.

CD200-positive cancer associated fibroblasts augment the sensitivity of Epidermal Growth Factor Receptor mutation-positive lung adenocarcinomas to EGFR Tyrosine kinase inhibitors.

Cancer associated fibroblasts (CAFs) play important roles in the chemotherapeutic process, especially through influencing the resistance of tumor cells to molecular targeted therapy. Here we report the existence of a special subpopulation of patient-specific-CAFs that augment the sensitivity of EGFR gene mutation-positive lung cancer to the EGFR-tyrosine kinase inhibitor (EGFR-TKI), gefitinib. When cocultured with EGFR mutation positive lung cancer cells, these CAFs increased the apoptic effect of gefitinib on cancer cells, whereas, in the absence of gefitinib, they did not affect cancer cell viability. The assay using different single cell-derived clones demonstrated that the aforementioned sensitizing ability is clone-specific. Microarray analysis revealed that CD200 was expressed at much higher levels in this CAFs. Knocking down of CD200 expression deprived CAFs of their sensitizing potential, suggesting that CD200 is the functional molecule responsible for the effect. Immunohistochemical analysis of samples from patients receiving postoperative gefitinib treatment revealed that the individuals whose resected lung adenocarcinomas contained CD200-positive CAFs tended to have longer progression free survival of gefitinib when they recurred after surgery. These results suggest that CD200-positive CAFs can augment the sensitivity to EGFR-TKIs and may possess far reaching applications in the therapeutic use of EGFR-TKIs.

Fibroblast-led cancer cell invasion is activated by epithelial-mesenchymal transition through platelet-derived growth factor BB secretion of lung adenocarcinoma.

Cancer-associated fibroblast (CAF)-dependent local invasion is the process by which cancer cells invade the extracellular matrix using tracks that have been physically remodeled by CAFs. In the present study, we investigated the process by which the epithelial-mesenchymal transition (EMT) of cancer cells affect CAF-dependent local invasion. Using an in vitro collagen invasion assay, we showed cancer cells undergoing EMT to promote the matrix-remodeling ability of CAFs and thereby enhance CAF-dependent local cancer cell invasion. Platelet-derived growth factor (PDGF)-BB secretion was significantly elevated in cancer cells undergoing EMT, and this induced an increase in the invasion ability of both CAFs and cancer cells. Conversely, knockdown of PDGF-B expression in cancer cells undergoing EMT, or treatment with a PDGF-receptor inhibitor, decreased the invasion ability of both CAFs and cancer cells. By analyzing the gene expression profiles of 442 patients with lung adenocarcinomas, we established that high expression of PDGF-B and presentation of mesenchymal-like tumors were significantly associated with a high rate of disease recurrence and poor patient prognosis. Thus, cancer cells undergoing EMT may accelerate their own ability to invade local tissues via PDGF-BB secretion to promote CAF matrix remodeling. Therefore, targeting PDGF signaling between cancer cells undergoing EMT and CAFs is a promising therapeutic target to inhibit cancer progression and improve patient prognosis.

Clonal heterogeneity in osteogenic potential of lung cancer-associated fibroblasts: promotional effect of osteogenic progenitor cells on cancer cell migration.

BACKGROUND: Cancer-associated fibroblasts (CAFs) consist of heterogeneous cell population in terms of their differentiation potential. The functional differences in tumor progression between CAFs with mesenchymal stem/progenitor cells (MSCs/MPCs) characteristics and CAFs without MSCs/MPCs characteristics are not clarified. METHODS: CAFs and vascular adventitial fibroblasts (VAFs, which contain MSCs/MPCs) were isolated from nine primary lung cancers and were cultured in osteogenic or adipogenic medium to assess their multi-lineage differentiation. Next, we established nine single-cell-derived clones from the primary culture of CAFs and examined their differentiation potential. The effects of each single-cell-derived clone on the proliferation and migration of lung adenocarcinoma cell line, A549, were analyzed. RESULTS: The nine samples of VAFs and CAFs showed various degrees of osteogenic differentiation. Although the VAFs displayed the ability to undergo adipogenic differentiation, all cases of the CAFs did not. CAFs clones presented varying degrees of osteogenic differentiation. Four clones displayed comparable levels of osteogenic potential with that of the VAFs, and two clones were completely negative. As compared to the CAFs clones that possessed lower osteogenic potential, CAFs clones with higher osteogenic potential did not confer proliferative activity in A549 cells. On the contrary, these clones significantly promoted the migration of A549 cells as compared to the clones with lower osteogenic potential. CONCLUSION: Our studies clearly indicate that CAFs derived from lung cancer are heterogeneous population that consists of cells with varying osteogenic potentials and that CAFs with higher osteogenic potential have a greater tumor-promoting function through the enhancement of cancer cell migration.