Table 2 Summary characteristics of included studies

Álvarez-Teijeiro, et al. [12]

2017

Spain

In vitro (Human)

head and neck squamous cell carcinomas (HNSCC)

1. Case sample: Fresh frozen surgical tissue specimens from 19 patients with HNSCC. Control sample: Normal epithelia from non-oncologic patients not exposed to tobacco carcinogens (e.g., childhood tonsillectomy)

Case sample: Formalin-fixed paraffin-embedded (FFPE) tissue samples from 40 patients diagnosed with laryngeal dysplasia. Control sample: FFPE normal epithelium

3. Case sample: Saliva samples were taken in advance from 15 patients with head and neck squamous cell carcinoma. Control sample: Saliva samples were collected from 11 healthy donors

1. Analysis of miRNA expression

2. miRNA target analysis using real-time RT-PCR

3. Cell Culture

4. Transfections using pre-miR miRNA precursors 5. Proteome Array and Western blot Analysis

6. Cell proliferation tests

7. Scratch-Induced Directional Migration Assay

8. Group three-dimensional spheroid invasion tests

9. Data Analysis

• miR-196a and miR-196b were shown to uniquely influence target genes and regulatory pathways in cells, affecting cell proliferation, migration, and invasion

• miR-196b dysregulation is an early event in HNSCC tumor formation, indicating its potential for early detection, disease monitoring, and use as a non-invasive biomarker in saliva

• The diverse impacts of miR-196a/b on distinct subcategories of HNSCC cells and neighboring CAFs might hinder the practicality of therapeutic interventions

Khazaei et al. [7]

2017

Iran

In vitro (Human)

esophageal squamous cell carcinoma (ESCC)

Case sample: We gathered 26 sets of fresh-frozen tissues and 39 blood serum samples from patients diagnosed with esophageal squamous cell carcinoma (ESCC) at Sayad Shirazi Hospital, part of Golestan University of Medical Sciences in Iran

The KYSE-30 cell line (human ESCC) and HFSF-PI3 cell line (human normal fibroblasts from skin) were obtained from the National Cell Bank of Iran (Pasteur Institute)

1. Isolation of RNA from esophageal tissue, serum samples, and purified exosomes

Quantification of miR-451 using quantitative reverse transcription polymerase chain reaction (RT-PCR)

3. Exosome isolation

4. Cell culture conditions and miR-451 overexpression

5. Co-cultivation of an esophageal cancer cell line with normal fibroblasts (HFSF-P13)

6. Migration Analysis

Analyzing MIF expression in the co-cultured cell lines

8. Statistical analyses

• Serum samples from patients with esophageal cancer exhibited elevated levels of miR-451. Subsequent analysis of cryopreserved tumor tissues from the same patients revealed reduced miR-451 expression

• When the KYSE-30 cell line was co-cultured with normal fibroblasts, there was a notable increase in the release of miR-451 exosomes into the culture medium. When the KYSE-30 cell line was co-cultured with fibroblasts expressing high amounts of miR-451, there was a significant increase in the migratory capacity of the KYSE-30 cells. Line

• The MIF expression in the KYSE-30 cell line showed an increase, although it was not statistically significant. MIF is a confirmed target of miR-451

• The study shows that cancer-associated fibroblasts use exosomal miR-451 as a signaling molecule to promote tumor cell migration and accelerate cancer progression

Chunping Wu1 – Et al [13]

2022

China

In vitro (Human)

supraglottic laryngeal squamous cell carcinoma (SLSCC)

Case sample:

Ten tumor specimens from 10 male patients with SLSCC

1. Primary culture of tumor specimens and corresponding surrounding connective tissues

2. Morphological analysis and immunocytochemical labeling of cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs)

3. Harvesting culture supernatant and isolating exosomes

4. Utilizing transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA)

5. Immunoblotting

6. Total RNA extraction

7. Analysis of small RNA (sRNA) sequencing and differential expression

8. Real-time quantitative polymerase chain reaction (RT qPCR) confirmation of specific miRNAs

9. Identification of potential exosomal miRNAs and their target genes

10. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)

11. Statistical analysis

• Exosomal miRNAs secreted by cancer-associated fibroblasts (CAFs) exhibited reduced expression levels, such as miR-656-3p, miR-337-5p, miR-29a-3p, and miR-655-3p. Conversely, miR-184-3p, miR-92a-1-5p, miR-212-3p, and miR-3135b exhibited elevated expression levels

• The KEGG analysis identified 30 crucial pathways associated with cancer start, progression, and cell cycle regulation

• The interaction network identified miR-16-5p, miR-29a-3p, miR-34c-5p, miR-32-5p, and miR-490-5p as the top five miRNAs, and CCND1, CDKN1B, CDK6, PTEN, and FOS as the top five target genes

• SLSCC patients exhibited unusual expression of miRNAs originating from exosomes of cancer-associated fibroblasts

• Identifying the top five miRNAs and their target genes may suggest an unfavorable tumor environment and act as therapy indicators for SLSCC

Shen et al. [4]

2017

China

In vitro

(Human)

Head and neck cancer (HNC)

Case example: 1. Cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) were obtained from patients who had surgery for head and neck cancers at the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine

The Dental School of the University of Maryland generously supplied HN13 cells

1. Establishing primary cell cultures of cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs)

2. Culturing cells from head and neck cancer

3. Immunoblotting

4. Immunofluorescence and immunohistochemistry

5. Cell cycle assessment

6. Analysis of miRNA array

7. Real-time PCR test

8. Migration test

9. Co-culturing of Head and Neck Cancer (HNC) cells with Cancer-Associated Fibroblasts (CAFs) or Normal Fibroblasts (NFs)

Construction of the RASSF2 luciferase reporter gene vectors

11. Data analysis

• miR-7 expression was shown to be increased in cancer-associated fibroblasts (CAFs)

• Increased miR-7 levels reduced RASSF2 gene expression, reducing PAR-4 release in the cancer microenvironment

• Cancer-associated fibroblasts (CAFs) reducing the release of PAR-4 may lead to enhanced proliferation and migration of cancer cells in the tumor microenvironment

• Targeting the disruption of the RASSF2-PAR-4 axis by miR-7 might be an innovative approach for gene therapy in head and neck cancers, as shown by the results

Min et al. [14]

2016

China

In vitro (human)

oral squamous cell carcinoma (OSCC)

Example case:

1. Nucleated fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were extracted from oral squamous cell carcinoma (OSCC) tumor tissues and identified based on specific markers

2. The human oral cancer SCC-25 cells were acquired from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences

1. Cell Line and Culture

2. Plasmids and Cellular Transfection

3. Real-Time PCR Analysis

4. Western Blot Analysis

5. Conduct Luciferase Reporter Assay

6. WNT10B Transfection and Immunodepletion

7. Cell Invasion and Migration Assay

8. Data Analysis

• Expression of miR-7 was shown to be elevated in cancer-associated fibroblasts (CAFs)

• Elevated miR-7 levels suppressed the expression of the target gene RASSF2, resulting in decreased PAR-4 release in the cancer microenvironment

• Cancer-associated fibroblasts (CAFs) decreasing the secretion of PAR-4 might result in increased cancer cell proliferation and movement within the tumor microenvironment

• miR-148a expression was decreased in cancer-associated fibroblasts (CAFs) compared to normal fibroblasts obtained from clinical oral squamous cell carcinoma (OSCC) tissue

• Increasing miR-148a levels in cancer-associated fibroblasts (CAFs) considerably hindered the movement and penetration of oral carcinoma cells (SCC-25) by focusing on WNT10B

The findings suggest that miR-148a may be a promising target for treating OSCC

Nouraee et al. [15]

2016

Iran

In vitro (human)

esophageal squamous cell carcinoma (ESCC)

Case sample: The esophageal squamous cell carcinoma cell line, KYSE-30, and the human regular fibroblasts cell line, HFSF-PI3, were acquired from the National Cell Bank of Iran at the Pasteur Institute in Tehran

1. Cell lines and co-culture system

2. Preparation of conditioned medium (CM)

3. Isolation of RNA and synthesis of cDNA

4. Amplification and profiling of MicroRNA

5. Analysis of data processing and pathway enrichment

Purification of exosomes via ultracentrifugation

Perform quantitative real-time polymerase chain reaction (qRT-PCR) on exosomal materials

• Discovered 18 miRNAs that exhibited substantial overexpression or downregulation in the conditioned medium of the co-culture system

• Pathways related to cell adhesion, endocytosis, and cell junctions may be connected to the CAF phenotype and tumor progression

• High amounts of miR-33a and miR-326 were found in the exosomes recovered from a conditioned medium of co-cultured and untreated cells

Their research clarified the role of cancer-associated fibroblasts (CAFs) in the tumor microenvironment through the secretion of miRNAs, proposing novel therapy options for esophageal and other types of cancer

LI‑PING SUN- et al. [1]

2019

China

In vitro

(Human)

Oral squamous cell carcinoma (OSCC)

Case example: 1. Participants and tissue specimens Forty-seven patients diagnosed with oral squamous cell carcinoma (OSCC), including 27 men and 20 women aged 39 to 72 years, who underwent tumor removal surgery at Liaocheng People's Hospital's Department of Oral Maxillofacial Surgery from January 1, 2014, to December 31, 2017

2. Culturing cells Oral squamous cell carcinoma The CAL-27 cells were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA)

1. Culturing cells

2. Fibroblast isolation 3. OSCC cells co-cultured with CAFs or NFs

4. Immunohistochemistry (IHC)

5. Immunocytochemistry

6. Isolation of exosomes

7. Conducting Western blot analysis

8. Transfection

9. Isolation of RNA and quantitative real-time PCR

10. Conducting Transwell migration and invasion tests

11. Analysis using flow cytometry

Identifying potential targets of miR-382-5p in Targetscan

13. Data analysis

• miR-382-5p levels were higher in cancer-associated fibroblasts (CAFs) than in fibroblasts from nearby healthy tissue. This increase in miR-382-5p was associated with the migration and invasion of oral squamous cell carcinoma (OSCC) cells

• It was shown that exosomes from cancer-associated fibroblasts carried miR-382-5p to oral squamous cell carcinoma cells

Exosomes derived from cancer-associated fibroblasts transfer miR-382-5p to oral squamous cell carcinoma cells, enhancing their ability to move and invade. The work validated a novel mechanism via which cancer-associated fibroblasts (CAFs) promote oral squamous cell carcinoma (OSCC) proliferation, providing possible avenues for cancer therapy

Junior et al. [6]

2021

Brazil

In vitro (human)

squamous cell carcinoma of the oral cavity (OSCC)

Case sample: We selected 26 cases and collected remaining FFPE blocks containing oral cavity squamous cell carcinoma (SCC) samples from patients who underwent primary curative resection at the Head and Neck Surgery Service of the Cancer Institute of the State of São Paulo (ICESP) in São Paulo, Brazil, between December 2010 and October 2015

Control sample: Tissues from the oral mucosa preserved in FFPE blocks were taken from 11 individuals who underwent operations for benign oral cavity disorders, forming the control group

1. Technique for detecting MicroRNA

2. Immunohistochemical analysis

3. Data analysis

• Elevated levels of miR-21-5p and miR-106-5p, together with decreased levels of miR-320a and miR-222-3p, suggested malignancy

• When examined separately, miR-21-5p had the best statistical performance in differentiating between tumor tissue and healthy mucosa

This work demonstrates the influence of microRNAs on the progression of OSCC and the notable alterations they induce in the tumor microenvironment

Saroj et al [16]

2021

Norway

In vitro (human)

oral squamous cell carcinoma (OSCC)

Case study:

Before any treatment, CAFs were extracted from biopsies of OSCC primary lesions from 13 patients at Haukeland University Hospital in Bergen, Norway

acquired matched normal oral fibroblasts (NOFs) from biopsies of cancer-free areas of the oral mucosa from five OSCC patients

Two OSCC cell lines, UK1 and Luc4, were used

Control samples were taken from biopsies of healthy volunteers' normal oral mucosa without cancer (n = 9)

1. Fibroblast isolation and cell culture

2. Extraction of total RNA and enrichment of small RNA for miR microarray analysis

3. miR Microarray

4. Transcription Reversal

5. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

6. Analysis of TCGA Data

7. Identification of miRNA Targets and Dual Luciferase Target Reporter Assay for miRNAs

8. Modulation of miR-204 in Cultured Fibroblasts

9. Isolation and Quantification of Proteins

10. Immunoblotting

11. 2D Co-Culture Migration Assay 12. Collagen Contraction Assay

13. 3D Organotypic Co-Cultures

Quantifying the invasion of oral squamous cell carcinoma (OSCC) cells in 3D-organotypic models

15. Data Analysis

• Modifying miR-204 expression did not affect fibroblast cell proliferation but did induce changes in cell motility properties, the concentrations of several molecules linked to cell motility, and the invasion of adjacent OSCC cells. Experimental validation using a 3′ UTR miR target reporter assay demonstrated that ITGA11 is directly targeted by miR-204

The study identified certain miRs with varying expression levels in stromal fibroblasts of OSCC lesions in comparison to normal oral mucosa. One of the miRs significantly decreased in cancer-associated fibroblasts (CAFs), miR-204, hinders the movement of fibroblasts and contributes to the suppression of malignancies. This is achieved by regulating the synthesis of several molecules and specifically targeting ITGA11

Matos et al. [17]

2020

China

In vitro (human)

Oral squamous cell carcinoma (OSCC)

Case example: 26 patients were chosen and categorized into two groups: 1. The superficial tumor group consisted of 12 neoplasms with a DOI of 5 mm. 2. The deep tumor group comprised 14 neoplastic patients with a DOI of 15 and 25 mm

Control sample: Eleven individuals with similar epidemiological characteristics gave healthy oral mucosa samples collected from oral surgery for benign mouth diseases, such as mucoceles or fibromas of the mucosa

1. MicroRNA detection technique

2. Immunohistochemical analysis

3. Statistical analysis

• miR-1-3p, miR-133-3p, and miR-21-5p showed varying expression levels in the superficial and deep tumor categories

• miR-21-5p had the best accuracy in distinguishing between superficial and deep cancers. This method was linked to decreased miR-1-3p levels and increased miR-21-5p levels

• Patients with elevated levels of miR-133a-3p had improved overall and disease-free survival rates

• The study demonstrated the correlation between microRNAs and the restructuring of the extracellular matrix in oral squamous cell cancer

• Deep OSCC specimens exhibited decreased levels of miR-1-3p and miR-133a-3p, together with increased levels of miR-21-5p, indicating potential involvement of these miRNAs in OSCC tumor invasion compared to superficial tumors

• Patients with elevated miR-133a-3p levels had better overall and disease-free survival rates, whereas miR-1-3p expression was linked to greater disease-free survival

Chen et al. [5]

2023

China

In vitro

(Human)

and

In vivo

(animal)

esophageal squamous cell carcinoma

(escc)

Ex vivo Case sample: ESCC tissues surgically excised were gathered for analysis. Six fresh and 53 paraffin-embedded samples of ESCC tissue without necrosis were chosen

Control sample: six fresh and 25 paraffin-embedded normal esophagus tissue samples

Twenty-five female BALB/c nude mice were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. for in vivo experiments

1. Hematoxylin–eosin (HE) staining and immunohistochemistry

2. Cell culture

3. Fibroblast extraction

4. Western blotting 5. Immunofluorescence staining

6. Exosome purification

7. Transmission electron microscopy

8. Analysis of particles using NanoSight particle tracking

9. Cultivation of Human Lens Epithelial Cells in a Prepared Growth Environment

10. Labeling and tracking exosomes

11. Cell migration analysis

12. Cell invasion assay

13. Cell proliferation analysis

14. Cell tube formation assay

15. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) 16. Gene transfection

Dual-luciferase reporter assay

18. Fluorescence in situ hybridization

19. Mouse models of tumors and their treatment

20. Statistical analysis

• Cancer-associated fibroblast-derived exosomes promoted lymphatic endothelial cells' proliferation, migration, invasion, and tube formation. Additionally, they stimulated lymphangiogenesis in ESCC xenografts

• Levels of miR-100-5p were much lower in exosomes from cancer-associated fibroblasts than in exosomes from normal fibroblasts

• miR-100-5p inhibited the proliferation, migration, invasion, and angiogenic potential of TLECs. Additionally, miR-100-5p inhibited the development of lymphatic vessels in ESCC xenografts

Exosomes derived from cancer-associated fibroblasts with decreased miR-100-5p levels can enhance lymphatic channel development, suggesting a possible strategy to inhibit lymphatic dissemination in esophageal squamous cell carcinoma by focusing on the IGF1R/PI3K/AKT pathway

Koji Tanaka – et al. [18]

2015

Japan

in vivo

(human)

and

In vitro

(human)

esophageal squamous cell carcinoma

(escc)

An in vivo case The study involved 64 patients who received neoadjuvant chemotherapy and underwent surgery for primary esophageal squamous cell carcinoma at the Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University

In vivo control sample: 27 healthy participants

Sample case in vitro: The verified human esophageal squamous cell line TE10 was acquired from the Riken Bioresource Center Cell Bank in Tsukuba, Japan

1. Assessment of the reaction to chemotherapy

2. Isolation and characterization of fibroblasts 3. Cell lines and culture conditions

4. RNA isolation

5. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for measuring miRNA levels

6. Quantitative real-time polymerase chain reaction

7. miRNA transfection

8. microRNA array

9. Immunofluorescence staining

10. Conducting growth inhibition experiments using cisplatin therapy and apoptosis assay

11. ELISA

12. Western blotting

13. Statistical analysis

• Increased levels of miR-27a/b expression correlated with a poor response to chemotherapy in patients with esophageal cancer

• Introducing miR-27a/b into cancer cells did not substantially impact their reaction to chemotherapy

• Esophageal cancer cells cultured in the medium surrounding normal fibroblasts transfected with miR-27a/b exhibited reduced responsiveness to cisplatin compared to cells cultured in the medium surrounding normal fibroblasts

• Transfected normal fibroblasts with MiR-27a/b showed elevated α-smooth muscle actin (α-SMA) expression, a characteristic of cancer-associated fibroblasts (CAF), and increased levels of transforming growth factor-β (TGF-β)

miR-27a/b contributes to chemotherapy resistance in esophageal cancer by inducing the conversion of normal fibroblasts into cancer-associated fibroblasts (CAFs)

Qitai Zhao – et al. [3]

2021

China

In vitro

(human)

and

In vivo

(Human)

esophageal squamous cell carcinoma (ESCC)

Case sample: Tumor specimens and peripheral blood were collected from patients with esophageal squamous cell carcinoma (ESCC) at the First Affiliated Hospital of Zhengzhou University. Peripheral blood was collected from individuals with esophageal squamous cell carcinoma who had four rounds of cisplatin-based treatment

The regular skin fibroblast cell line (HSF-1) and tumor cell lines (EC1 and KYSE70) were acquired from the Chinese Academy of Sciences Cell Repertoire in Shanghai, China

1. In vitro cell cultivation

2. Isolation and cultivation of primary cancer-associated fibroblasts. 3. Co-culture study

4. Conduct Western blot analysis

5. Flow cytometry analysis

6. Isolation and purification of exosomes

7. Exosome identification

8. Analysis of Exosome Uptake

9. Analysis using immunohistochemistry

10. Immunofluorescence staining

11. Real-time PCR

Transfection assay using miR-21 mimics and inhibitors

13. Analysis of survival

14. Analysis with Cytometric Bead Array

15. Analysis of bioinformatics

16. Statistical analysis

• Research has shown that IL-6 and exo-miR-21, produced by cancer-associated fibroblasts, converted monocytes into monocytic-myeloid-derived suppressor cells by collectively activating the STAT3 signaling pathway

• Elevated levels of M-MDSCs and CAFs in tumor tissue correlated with resistance to DDP and a reduced estimated overall survival in patients with ESCC

• Cancer-associated fibroblasts release exosomes containing miR-21 which are transferred to monocytes. Exo-miR-21 is released into the cytoplasm and targets PTEN

• IL-6 is acknowledged as the primary cytokine responsible for activating STAT3. MiR-21 stimulates STAT3 by targeting PTEN in coordination with IL-6. IL-6 and exo-miR-21 were discovered to enhance STAT3 phosphorylation, leading to the conversion of monocytes into M-MDSCs

• Cancer-associated fibroblasts produced myeloid-derived suppressor cells that shielded tumor cells from cisplatin-induced cell death, resulting in unfavorable outcomes for patients with esophageal squamous cell carcinoma

This highlights the crucial partnership between cancer-associated fibroblasts and myeloid-derived suppressor cells in enhancing medication resistance

Guiquan Zhu – Et al [19]

2021

china

In vitro

(human)

and

In vivo

(animal)

head and neck squamous cell carcinoma (HNSCC)

Two human HNSCC cell lines, SCC-9 and CAL-27, were acquired from and verified by ATCC for in vitro experimentation

MRC-5 cells were acquired from the China Center for Type Culture Collection

Female Nude mice were acquired from the Laboratory Animal Center of Sichuan University in Chengdu, Sichuan, China, for the in vivo case sample

1. Cell culture under hypoxic conditions

Isolation of small extracellular vesicles (sEVs) and extraction of RNA

3. Scanning electron microscopy

4. Construction and sequencing of miRNA library

5. Quantitative real-time PCR (qRT-PCR)

6. Immunofluorescence (IF) and immunohistochemistry (IHC)

7. Immunoblotting

8. Invasion analysis

9. Extracellular acidification rate (ECAR)

10. Utilization of cytokine array and Enzyme-Linked Immunosorbent Assay (ELISA)

Xenograft

12. ChIP assay

13. Data analysis

• Hypoxic head and neck squamous cell carcinoma cells induced the conversion of cancer-associated fibroblasts by releasing transforming growth factor beta (TGF-β) and small extracellular vesicles (sEVs) harboring elevated levels of miR-192/215 family microRNAs

• Caveolin-1 (CAV1), a gene controlled by miR-192/215, inhibited the TGF-β/SMAD signaling pathway and promoted the transformation of fibroblasts into cancer-associated fibroblasts (CAFs). High levels of miR-192/215 in small extracellular vesicles (sEVs) from Head and Neck Squamous Cell Carcinoma (HNSCC) tissue, as opposed to blood serum, indicate the existence of hypoxic and aggressive malignant tissue

• miR-215 was found in small extracellular vesicles (sEVs) from tumor tissue but not in circulating sEVs. It was linked to a worse overall survival rate in patients with head and neck squamous cell carcinoma (HNSCC)

Small extracellular vesicles (sEVs) carry miRNAs from cancer cells to fibroblasts, aiding in remodeling the hypoxic tumor microenvironment

• Extracellular vesicles that are soluble and derived from cancerous tissue may serve as a potential source of biomarkers.

Hypoxia increases the concentrations of miR-192/215 in the small extracellular vesicles (sEVs). Fibroblasts absorb small extracellular vesicles (sEVs) with elevated miR-192/215 levels, resulting in reduced CAV1 expression and activation of TGF-β signaling. The regulatory mechanism enables cancer cells to induce the conversion of stromal fibroblasts into cancer-associated fibroblasts, promoting the progression of tumor cells in a feedback loop. Small extracellular vesicles (sEVs) deliver microRNAs to cancer cells and stromal fibroblasts, aiding in the remodeling of the hypoxic tumor microenvironment in the Head and Neck Squamous Cell Carcinoma (HNSCC). Solid extracellular vesicles (sEVs) derived from cancer tissues, rather than peripheral blood, have the potential to serve as vehicles for biomarkers

Li et al. [11]

2017

China

In vitro (human)

and

In vivo (animal)

Oral squamous cell carcinoma (OSCC)

In vitro case sample:

1. SCC-9 and CAL-27 cells were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA)

2. Patient samples and cell isolation A total of 6 paired fresh, surgically resected OSCC tumors and matched non-malignant adjacent tissues were obtained from Chenzhou No.1 people's hospital

In vivo case sample:

Female BALB/c nude mice (4-week-old)

1. Cell Culture and Reagents

2. Patient samples and cell isolation

3. Transfection of cells with pre-miRNA precursors or anti-miRNA inhibitors

4. Lentivirus transduction and establishment of stable cell lines

5. Quantitative real-time PCR

6. DNA methylation analysis

7. Luciferase reporter assay

8. Cell migration assay

9. CCL2 and IL-8 ELISA assay

10. Mouse xenograft models

11. Statistical analysis

• DNA hypermethylation leads to the downregulation of miR-124 in cancer-associated fibroblasts (CAFs), oral cavity carcinomas (OCCs), and oral squamous cell carcinoma (OSCC) tissues. Moreover, miR-124 plays a vital role in controlling the communication between cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) and ovarian cancer cells (OCCs) via influencing the production of CCL2/IL-8

• Injecting miR-124 intravenously decreased oral tumor development in mice, indicating that increasing miR-124 levels might be a novel therapeutic strategy for treating oral cancer patients

miR 124 rescue could be a potential rationale for therapeutic applications in oral cancer

Li et al. [8]

2018

China

In vitro (human)

and

In vivo (animal)

oral squamous cell carcinoma (OSCC)

In vitro case sample:

1. Primary human CAFs and donor-matched NFs were isolated from OSCC patients treated by surgical resection at the Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University

2. Human oral keratinocytes (HOK) were purchased from ScienCell

3. OSCC cells, including CAL27 and SCC15, were kindly donated by the Department of Central Laboratory, Peking University, School and Hospital of Stomatology

In vivo case sample:

BALB/c nude mice (Beijing Vital River, Beijing, China) at 4–6 weeks of age were used

1. Isolation of primary human fibroblasts and OSCC cell culture

2. Exosome preparation

3. Preparation of lentiviral vector and transfection

4. Immunofluorescence

5. Quantitative Real-Time PCR

6. miRNA sequencing

7. Western blotting

8. Gelatin zymography

9. Functional assay

10. Luciferase reporter assay

11. In situ hybridization

12. Animal models

13. Statistical analysis

• The research showed a significant reduction in miR-34a-5p in exosomes from cancer-associated fibroblasts (CAFs) and proved that fibroblasts may transfer exosomal miR-34a-5p to oral squamous cell carcinoma (OSCC) cells

• In xenograft studies, it was shown that increasing the levels of miR-34a-5p in cancer-associated fibroblasts (CAFs) inhibited the development of tumors from oral squamous cell carcinoma (OSCC) cells

• MiR-34a-5p was shown to hinder the proliferation and dissemination of OSCC cells by attaching to its specific target, AXL

• Continuous abnormal synthesis of AXL in OSCC cells with increased levels of miR-34a-5p counteracted the decrease in cell growth and movement induced by the miRNA. The miR-34a-5p/AXL axis promoted the advancement of OSCC by activating the AKT/GSK-3β/β-catenin signaling pathway, resulting in the initiation of epithelial-mesenchymal transition (EMT) and boosting cancer cell metastasis. The miR-34a-5p/AXL pathway facilitated the translocation of β-catenin to the nucleus, resulting in enhanced SNAIL production through transcriptional upregulation. As a result, both MMP-2 and MMP-9 were activated

The miR-34a-5p/AXL axis confers aggressiveness in oral cancer cells through the AKT/GSK-3β/β-catenin/Snail signaling cascade and might represent a therapeutic target for OSCC

Qin, et al. [20, ]

2019

china

In vitro (human)

and

In vivo (animal)

head and neck cancer (HNC)

In vitro case sample:

1. 80 pairs of tumor and adjacent normal tissues were obtained from patients diagnosed with primary HNC and underwent initial surgery between September 2011 and June 2015; another 28 pairs were collected between July 2018 and September 2018

2. In another 40 HNC patients, plasma samples were collected one day before surgery and three days after tumor resection

3. SCC-4, SCC-9, SCC-25, CAL 27, and 293 T cells were purchased from the American Type Culture Collection (ATCC, USA), and the University of Maryland Dental School, USA kindly provided the human HNC cell lines HN4, HN6, and HN30

In vitro control sample:

Plasma samples from 30 donors who had undergone physical examination at the Ninth People’s Hospital were selected as healthy controls

In vivo case sample:

A tumor-bearing model was constructed in BALB/C athymic nude mice (4 weeks old)

1. Cell cultures

2. Immunofluorescence

3. Western blot analysis

4. MTT assay

5. CM preparation

6. Exosome isolation

7. Transmission electron microscopy

8. Fluorescent labeling and transfer of exosomes

9. Co-culture assay

10. Plasmid construction

11. Cell transfection

12. Biotin miRNA pull-down assay

13. RNA extraction and real-time PCR analysis

14. RIP assay

15. Luciferase analysis

16. Immunoprecipitation of miRNA targets

17. Tumorigenicity assay in vivo

18. Statistical analyses

• Cancer-associated fibroblasts (CAFs) are naturally resistant to cisplatin and play a crucial role in controlling the survival and growth of head and neck cancer cells by transferring active miR-196a from CAFs to tumor cells through exosomes

• Exosomal miR-196a interacts with new targets, CDKN1B and ING5, to provide resistance to cisplatin in head and neck cancer cells. Eliminating exosome or exosomal miR-196a from cancer-associated fibroblasts (CAFs) restored head and neck cancer (HNC) sensitivity to cisplatin

• Cancer-associated fibroblast-derived exosomes may employ heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) to assist in packaging miR-196a

• Higher levels of plasma exosomal miR-196a are associated with reduced overall survival rates and resistance to therapy

The study discovered that miR-196a from CAF-derived exosomes causes resistance to cisplatin in head and neck cancer via affecting CDKN1B and ING5. This suggests that miR-196a might be a valuable indicator and a possible target for overcoming cisplatin resistance in head and neck cancer

Jin Yang1 – Et al [21]

2021

china

In vitro (human)

and

In vivo (animal)

oral squamous cell carcinoma (OSCC)

In vitro case sample:

1. 6 OSCC patients (were obtained from the West China Hospital of Stomatology at Sichuan University during 2017–2019; The OSCC patients were 45–63 years old, experienced no relapses, and underwent no preoperative chemotherapy and/or radiotherapy.)

2. The human OSCC cell lines Cal-27, UMSCC-1, HSC-2, and FaDu were purchased from ATCC or obtained from the State Key Laboratory of Oral Diseases

In vitro control sample:

1. 6 patients who received third molar extraction as normal controls

2. Cal-27 cells were used alone as the control group in this study

In vivo case sample:

Four- to six-week-old BALB/c nude mice, half male and half female, were purchased from Charles River (Beijing, China)

1. Clinical tissue sample collection and primary cell culture

2. Plasmid construction and cell transfection

3. Cell lines and mice

4. RNA isolation, RNA sequencing, and qRT-PCR

5. Immunohistochemistry and immunofluorescence

6. Histology and immunohistochemistry

7. Western blotting

8. RNA-FISH and luciferase reporter assays

9. Bioinformatic analysis

10. Statistical analysis

Using siRNA to knock down lncRNA H19 suppressed the MAPK signaling pathway and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and miR-675-5p. Additionally, the lncRNA H19/miR-675-5p/PFKFB3 axis was found to promote the glycolysis pathway in oral cancer-associated fibroblasts (CAFs), confirmed through luciferase reporter system assays and treatment with a specific miRNA inhibitor

miR-675-5p/PFKFB3 act as key regulators in lncRNA H19-mediated glycolysis in oral CAFs

The lncRNA H19 was found to be a crucial lncRNA in oral cancer-associated fibroblasts (CAFs) and was simultaneously increased in both oral cancer cell lines and CAFs. Using small interfering RNA (siRNA) techniques, we found that reducing lncRNA H19 levels impacted the growth, movement, and glucose metabolism of oral cancer-associated fibroblasts (CAFs). The study introduces a novel approach to analyzing glucose metabolism in oral cancer-associated fibroblasts (CAFs), potentially offering a unique biomarker for oral squamous cell carcinoma (OSCC) diagnosis and a fresh target for anti-tumor treatment

Jin et al. [21]

2021

China

In vitro (human)

and

In vivo

(animal)

Esophageal squamous cell carcinoma (ESCC)

In vitro case sample:

1. Samples of tumor tissue and precancerous tissue were collected from five patients who did not receive any preoperative chemotherapy or radiotherapy enrolled in the Cancer Institute and Hospital, Guangzhou Medical University (Guangzhou, China)

2. Patient-derived ESCC cell lines, human ESCC cell lines (EC-18 and KYSE30), and embryonic kidney cell line 293 (HEK293) were provided by Professor Guan from the Department of Clinical Oncology, The University of Hong Kong

In vivo case sample:

Four- to six-week-old female Balb/c mice were used to examine allograft tumor growth

1. Cell Culture

2. Isolation and Quantification of Exosomes

3. Cell migration, colony formation, and wound healing assays

4. Fluorescent immunostaining

5.MicroRNA sequencing

6. Quantitative real-time PCR

7. Western blot analysis

8. Animal experiments and welfare

9. Statistical analysis

• The mRNA level of hsa-miR-3656 showed a significant rise in these exosomes

• Comparisons between tumor cell behavior in lab dishes and animals showed that the presence of miR-3656 significantly improved the proliferation, migration, and invasion capacities of esophageal squamous cell carcinoma (ESCC) cells

• ACAP2 was discovered as a target gene controlled by miR-3656, detrimental to tumor development. Exosome-delivered miR-3656 suppresses ACAP2, leading to activation of the PI3K/AKT and β-catenin signaling pathways, promoting ESCC cell proliferation in laboratory settings and xenograft models

These findings indicate that exosomes carrying miR-3656 from cancer-associated fibroblasts (CAFs) play a role in the aggressive advancement of esophageal squamous cell carcinoma (ESCC). These results suggest a promising therapeutic target for ESCC and offer a fresh perspective for clinical treatment strategies

Li, et al. [22]

2022

China

In vitro

(human)

and

In vivo

(animal)

Nasopharyngeal carcinoma (NPC)

In vitro case sample:

1. Tumor tissues from thirty patients diagnosed with NPC by biopsy from January 2016 to June 2017 were collected at the Hainan Affiliated Hospital of Hainan Medical University

2. Human NPC cell lines CNE2, HNE1, HNE2, HONE1, C666-1, SUNE-1, nasopharyngeal epithelial NP69 cells, and 293 T cells provided by the National Collection of Authenticated Cell Cultures

In vitro control sample:

Thirty normal nasopharyngeal tissues

In vivo case sample:

Eight- to ten-week-old male BALB/c nude mice

1.Cell culture and treatment

2.Cell transfection

3.Conditioned medium (CM) preparation

4.Exosome isolation and characterization

5.Iodixanol density gradient centrifugation

6.Real-time quantitative reverse-transcription PCR (RT-qPCR)

7.Western blot

8.Cell Counting Kit-8 (CCK-8) assay

9.Colony formation

10.Wound healing assay

11.Transwell assay

12.Ubiquitination analysis of TRIM24

13.Dual-luciferase reporter assay

14.Co-Immunoprecipitation (Co-IP)

15.RNA immunoprecipitation (RIP)

16.Chromatin immunoprecipitation (ChIP)

17.A xenograft mouse model of NPC

18.Statistical analysis

• Molecules such as MiR-106a-5p, TRIM24, and SRGN were shown to be upregulated in both NPC tissues and cells, but FBXW7 was downregulated

• Exosomes containing miR-106a-5p were able to enter NPC cells. High levels of miR-106a-5p appeared to enhance the proliferation, migration, invasion, and metastasis of cancer cells. Scientists saw a decrease in aggressive behaviors when they lowered the amounts of this miRNA

• miR-106a-5p targets FBXW7, a protein that regulates the levels of TRIM24, another factor in this cancer scenario. TRIM24 influences SRGN, a protein crucial for cancer development

• Researchers successfully inhibited miR-106a-5p in exosomes, decelerating the development and metastasis of NPC in live creatures during studies. It appears that a small chemical, transported by exosomes, has a significant role in increasing the aggressiveness of NPC

Exosomal miR-106a-5p accelerates the progression of NPC by decreasing FBXW7 expression, increasing TRIM24 levels, and elevating SRGN expression. The research illuminates the role of exosomal miR-106a-5p on NPC's proliferation and suggests potential treatment strategies by targeting these pathways

Wei‑Zhou Wang – Et al [10]

2023

china

In vitro (human)

and

In vivo (animal)

oral squamous cell carcinoma (OSCC)

In vitro case sample:

1. hOMF (CellResearchCorp, Singapore)

2. Cal-27 (Cellcook, Guangzhou, China)

In vivo case sample:

BALB/c-Nude mice were purchased from the Animal Experiment Center of Kunming Medical University

1. Induction of CAFs and extraction of CAFs-Exo

2. Cal-27 cells were co-cultured with CAFs-Exo or hOMF-Exo

3. Tumor-forming and exosomes injection in nude mice

4. Protein extraction and western blotting

5. Detection of Cal-27 and exosome genes with quantitative PCR assay

6. Sequencing and raw data processing

7. Identification of immune-associated genes

8. PPI network construction and module analysis

9. Screening of candidate genes related to immune cells

10. mRNA-miRNA interaction network construction by WGCNA

11. Development of immune-related signature

12. Analysis of CAFs-Exo target genes by mRNA-miRNA interaction network

13. Statistical analysis

• hsa-miR-139-5p and ACTR2 found in CAFs-Exo may have increased the amounts of CD81 and PIGR in Cal-27 cells, thereby clarifying CAFs-Exo's role in enhancing OSCC proliferation

• CAFs-Exo has a potent capacity to promote OSCC proliferation and are linked to immunological suppression. Through the analysis of sequencing data from CAFs-Exo and publically accessible TCGA data, it was found that immune-related genes in CAFs-Exo can impact the expression of PIGR, CD81, UACA, and PTTG1IP in Cal-27 cells. CAF-derived exosomes may be involved in immunological regulation and promoting the formation of oral squamous cell carcinoma

CAFs-Exo is involved in controlling tumor immunity through hsa-miR-139-5p, ACTR2, and EIF6. PIGR, CD81, UACA, and PTTG1IP are potential targets for future OSCC therapies