Oral cancer secretome induce the expression of a group of genes that control the vitamin D signaling pathway in T cells

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A collaborative research led by immunologist Estefania Nova-Lamperti from the Universidad de Concepción (Chile), with a branch of researchers from MELISA Institute and other international academic centers, made progress in the understanding of molecular mechanisms preventing an effective antitumor immune response in oral cancer; The latter due to the production of chemical mediators that induce an anti-inflammatory regulatory response that favors tumor development through the vitamin D signaling pathway.

Oral cancer, 90% of which corresponds to the squamous cell type, is a neoplasm with a high mortality and morbidity rate, mainly because the diagnosis is made in late stages when metastases already exist, and where treatment produces serious physical and functional sequel among survivors.

It is well known that the immune system plays a key role in the development of cancer, either by stimulating pathways that play an anti-tumor role or, conversely, by generating an anti-inflammatory environment that allows the tumor to grow and be spread.

The main biological agents of the immune system are lymphocytes or T cells, which have different functions or phenotypes. In cancer, the presence of regulatory T cells (Tregs) and helper T cells type 2 (Th2) are associated with a worse prognosis, whereas the responses of helper T cells type 1 (Th1) within tumors, in general, show a better prognosis.

Dr. Nova-Lamperti points out that a key question in oral cancer is how an anti-inflammatory microenvironment is induced, boosting tumors growth. To date, “the immunoregulatory mechanisms associated with this change in the immune response are unknown and it is not acknowledged whether they come from or are external to the tumor,” explains the immunologist.

Using flow cytometry techniques, the research team was able to feature the T-cell phenotypes predominant in biopsies of 15 patients with oral cancer and compared them with the T-cell populations found in biopsies of 16 disease-free controls. Thus, they identified a predominant distribution of T cells expressing CCR8+ receptors (generally abundant in skin tissues), Th2-like regulatory T cells, and a small population of Th1 cells.

Based on these new findings, the researchers hypothesized that the tumor microenvironment obtained from biopsy cultures of patients with oral cancer, which contains the factors secreted by tumor cells (known as secretome), had the ability to induce an anti-inflammatory phenotype itself.

To test this assumption, Nova-Lamperti and her colleagues challenged immune T cells subpopulations with the cancer secretome and, using genomics and proteomics techniques, determined how the mRNA transcripts and proteins expressed by these cells are modified.

Notably, transcriptomics showed that oral cancer secretome induced the expression of a group of genes that control the vitamin D (VitD) signaling pathway in T cells.

Moreover, the proteomics study, through high-resolution mass spectrometry, revealed the presence of several proteins associated with the production of prostaglandin E2 (PGE2) linked to VitD rapid signaling in cell membranes. In addition, the researchers found a reduction in the proteins that carry VitD into the cell.

Based on these findings, the researchers suggested that the decrease in the mobility of VitD promotes an increase in its concentration in the tumor microenvironment, inducing an anti-inflammatory phenotype favorable to the tumor. In new experiments, the researchers challenged T-cell cultures with VitD and PEG2, confirming that VitD induces a Th2 regulatory response with expression of CCR8, while the combination of VitD and PEG2 inhibited the production of small proteins called cytokines.

The latter are important for the activation of the immune system. Additionally, the study showed that a cytokine that binds to the CCR8 receptor, known as chemokine CCL18, was overexpressed in tumors, favoring a vicious circle that stimulates the Tregs cells to ‘stagnate’ in the tumor microenvironment.

Regarding the effect of the study, the immunologist noted that “findings could be interesting for the development of biological therapies centered on antibodies capable of blocking the action of specific molecules that favor tumor growth. For example, in the case of oral cancer, a therapy that selectively blocks the CCR8 receptor or that normalizes the vitamin D signaling pathway could eventually slow tumor growth, decrease the sequel of surgical resections, and improve survival in these patients.” The researcher stressed the need to continue doing research in this regard.

Researcher Mauricio Hernández, a mass spectrometry expert at the MELISA Institute, stated that “collaborating in this research was challenging; the proteomics for this study required several time-consuming procedures to obtain optimal results. For example, because we dealt with cell culture media, each sample had to be depleted of abundant proteins such as albumin that ‘shield’ or make it difficult to detect smaller, less abundant proteins; then we performed an off-line fractionation to increase our identification capability.

This encompasses a chromatography step to subdivide each sample, which substantially multiplies the number of runs on our mass spectrometer and increases the time in subsequent bioinformatic analysis.”

Finally, Prof. Elard Koch, senior researcher and Chairman at the MELISA Institute, said that they were pleased to be invited to participate in the study led by Dr. Nova-Lamperti; “Collaborating with our multi omics capabilities in research as relevant as this one is encouraging for our researchers and a key goal for our institute” Koch remarked.


Oral mucosal cancer is cancer that arises from the lining (mucosa) of the oral cavity. The oral cavity is compromised of the mucosa lining of the lips and cheeks, the teeth, gingiva (gums), anterior two-thirds of the tongue, the floor of the mouth, hard palate, and the retromolar trigone posterior to the wisdom teeth. It has a close anatomical relationship with the oropharynx, the boundary of which is the border between the hard and soft palate, the border between the anterior 2/3rds and posterior 1/3rd of the tongue, and the anterior pillars of the tonsils. The main risk factors are smoking and alcohol consumption. The mainstay of treatment is surgery, often with adjuvant radiotherapy.

Etiology

Tobacco 

Smoking tobacco is the greatest risk factor and cause of oral cancer due to its carcinogenic chemicals, including nitrosamines, benzopyrenes, and aromatic amines.[1] The risk of developing oral cancer is 3 times higher in smokers compared with non-smokers.[2] Individuals are also at risk from secondary passive smoking environments.[3] Studies have shown a synergistic relationship with alcohol consumption, resulting in a higher risk of malignancy.

In various parts of the world, tobacco is chewed or held in the mouth rather than smoked.  Nicotine is absorbed via the mucous membranes to provide the desired effect. It is most closely linked to oral cavity cancers due to direct contact with tissues affected but is also associated with oropharyngeal malignancies. 

  • Chewing betel quid: also known as ‘pan’ or ‘paan,’ uses a combination of betel leaf, areca nut, slaked lime, and tobacco, which is then chewed. It is a widespread practice in Asia and is associated with an even higher risk of malignancy compared with smoking tobacco alone due to the prolonged exposure of the carcinogens to cells in the mouth.[2]
  • Snuff/Snus: a moist form of smokeless tobacco commonly placed under the upper lip for prolonged periods of time. This practice is most common in Scandinavia and North America.[4] 

Alcohol 

Consuming alcohol, particularly in conjunction with smoking, increases the risk of oral cancer. Despite no clear carcinogenic properties of ethanol itself, alcohol has been shown to increase the permeability of oral mucosa, thus enabling damage by other carcinogens.[5] 

HPV 

Human Papilloma Virus (HPV), mainly types 16 and 18, have been shown to be associated with malignancy. Although it is most closely associated with cervical cancer and oropharyngeal cancer (particularly tonsillar and base of tongue tumors), there is some evidence to suggest that there is an association with oral cancers. In the oral cavity, HPV infection is 4 times more likely in those with squamous cell carcinomas by comparison to healthy mucous membranes.[6] The spread of infection is mostly through oral sexual contact. 

Stem Cell Transplants 

Patients who have undergone hemopoietic stem cell transplants are 4 to 7 times more likely to develop oral cancer compared with the average population. Evidence of graft versus host disease in the oral cavity often precedes this. Symptoms include mucositis, xerostomia, and lichenoid changes. The tongue and salivary gland are the most common cancer sites developing 5 to 9 years after transplant.[7]

Epidemiology

It can be challenging to differentiate the incidence and prevalence of oral cavity cancers independently of oropharyngeal cancers as there is a huge variation in the categorization of these cancers. Many institutions will group data despite being distinct diseases, which can be confusing. According to the American Cancer Society’s Global Cancer Facts and Figures 4th Edition Report in 2018, the global incidence is reported as approximately 2% of all cancers. In males in medium human development (HDI) index countries, lip and oral cavity cancer (together with lung cancer) are the most commonly diagnosed cancers.

Although oropharyngeal cancer incidence is increasing (particularly HPV positive cancers), the incidence of oral cavity cancers is, in fact, decreasing. Countries in Southern Asia, such as India and Sri Lanka, and the Pacific Islands, have the highest incidence of oral cavity and lip cancers worldwide.[8] The incidence is approximately double in males versus females, likely due to the higher rate of carcinogenic activities such as smoking and alcohol consumption.

Histopathology

Squamous cell carcinomas account for over 90% of cancers of the oral cavity.[9] Premalignant lesions that display dysplasia such as erythroplakia and leukoplakia are associated with the development of squamous cell carcinomas. 

Other malignant types of tumors are listed below.  

  • Verrucous carcinoma 
  • Mucosal melanoma 
  • Kaposi sarcoma
  • Primary intraosseous squamous cell carcinoma
  • Osteosarcoma
  • Rare malignant tumors: e.g. fibrosarcoma, liposarcoma, lymphoma, chondrosarcoma, plasmasarcoma

History and Physical

Oral mucosal cancer presents clinically in various ways depending on its location. Early disease may manifest as irregular white, red, or mixed patches on the mucosa. More established cancers appear as an indurated raised nodule, often with an ulcerated surface that may cause little pain.  If cancer has spread locally or systemically, patients may present with dysphagia, odynophagia, hoarse voice, otalgia, weight loss, and lymphadenopathy.

A thorough clinical examination of the entire oral cavity is key in identifying potential tumors and spotting concurrent tumors and/or spread. Examination with two tongue depressors and a good light source should be carried out in addition to neck examination to assess for any lymphadenopathy. Regional neck lymphadenopathy is recorded according to the anatomical levels I to VI. A flexible nasendoscopy should be performed to check for any concurrent oropharyngeal or laryngeal tumors.

Evaluation

Biopsy 

Initial investigations include a tissue biopsy of the oral lesion. If tolerated and easily accessible many lesions can be biopsied in an outpatient clinic. Ultrasound-guided fine-needle aspiration (FNA) may be carried out if there is associated lymphadenopathy. For tongue base or more posterior lesions, an examination under general anesthetic (EUA) should be performed in order to obtain a tissue sample for histology.[9]

Endoscopy 

In addition to simple nasendoscopy in the clinic, panendoscopy should be performed under general anesthetic to look for concurrent tumors of the pharynx and larynx. 

Imaging 

Magnetic Resonance Imaging (MRI) is the modality of choice in evaluating the tumor itself, soft tissue involvement, and local perineural invasion. Computed tomography (CT) scans should be requested to assess the involvement of the bone, lymph nodes, and chest. Positron emission tomography (PET) scans may be used for assessment in cases where the location of the primary cancer is unclear.

Treatment / Management

Surgical

The mainstay of curative treatment usually involves surgical excision. The extent of surgery will depend on the size and staging of cancer but will usually involve a wide local excision. If there is a local invasion or lymph node spread, additional lymph node excision or neck dissection may be indicated or undertaken electively. Histological examination of excised tumors will be undertaken to ensure that there are clear margins. A temporary tracheostomy may be required in order to provide a safe airway following upper airway swelling from the operation. For more extensive resections, a free flap reconstruction may be required. 

Chemoradiotherapy 

Chemotherapy, radiotherapy, or both may be used in conjunction with surgery in order to eliminate any further malignant cells; this is usually done post-operatively. Its use is associated with multiple short and long-term side effects including xerostomia, nausea, dysphagia, mucositis, hair loss. 

Monoclonal Antibodies 

Cetuximab, an epidermal growth factor receptor (EGFR) inhibitor can also be used in conjunction with radiotherapy. This is usually used for locally advanced, recurrent, or metastatic disease.[10]

Palliation

For aggressive or advanced tumors or when a patient has other significant co-morbidities that precludes them from curative treatment, it may be felt that a palliative approach is in the patient’s best interest. This most often involves palliative radiotherapy, as well as anticipatory medications for symptom control and end of life care.

Differential Diagnosis

  • Pre-cancerous lesions: erythroplakia, leucoplakia 
  • Benign oral mucosal lesions: geographic tongue, median rhomboid glossitis, necrotizing sialometaplasia, hairy tongue, oral hairy leukoplakia, oral candidiasis, herpetic gingivostomatitis, aphthous ulcers, traumatic ulcers, herpes labialis
  • Benign tumors: papilloma, lipoma, lingual thyroid, mucocele, ranula, neurofibroma, haemangioma, oral keratoacanthoma 
  • Odontogenic tumors 

Staging

Staging 

Tumor, Node, Metastasis (TNM) staging is used to categorize tumors and aid prognostication as well as plan treatment. This is carried out using both clinical, histological, and radiological evaluation with attention to the primary tumor, lymph nodes in the neck, and distant metastases. In 2017, The American Joint Committee on Cancer published changes in the 8th edition Cancer Staging Manual on TNM classification for oral cavity and oropharyngeal cancers in order to better distinguish between the two cancer types.[11] 

T Classification

  • T1: size ≤2 cm and DOI ≤5 mm 
  • T2: size 2-4 cm and DOI ≤10 mm or size ≤2 cm and DOI 5-10 mm
  • T3: size >4 cm or any tumor>10 mm DOI
  • T4a: tumor invades through the cortical bone of the mandible or maxillary sinus, or invades the skin of the face
  • T4b: tumor invades the masticator space, pterygoid plates, or skull base, or encases internal carotid artery

N Classification 

Clinical 

  •  NX: regional node involvement cannot be assessed
  •  N0: no LN involved
  •  N1: single ipsilateral LN ≤3 cm 
  •  N2a: single ipsilateral LN, 3-≤6 cm 
  •  N2b: multiple ipsilateral LNs, all ≤6 cm
  •  N2c: any bilateral or contralateral LNs, all ≤6 cm

(all above no extra-nodal involvement ENE(-))

  •  N3a: size >6 cm and ENE(-)
  •  N3b: any ENE(+), either clinical or radiographic

Pathologic 

  • N1–N2: same as above and ENE(-) with exception of:
  • N2a includes lymph node ≤3 cm, ENE(+)
  • N3a size >6 cm and ENE(-)
  • N3b: ≥3 cm and ENE(+) LN or >1 ENE(+) LNs
  • M Classification 
  • M0 No distant metastases
  • M1 Distant metastases 
  • LN = lymph node 

DOI = depth of invasion 

ENE(+) = extra-nodal extension present 

ENE(-) = extra-nodal extension absent

Prognosis

Prognosis and survival rates depend on the staging of cancer at diagnosis, adequate and prompt treatment, and local expertise to deliver this. 5-year survival rates drop significantly in those whose disease has locally spread and even further if there are distant metastases, highlighting the importance of early diagnosis. The American Cancer Society estimates survival rates of oral and oropharyngeal cancers together. They state that for those with local disease, 5-year survival is around 84%. This drops to 66% and 39% with regional and distant spread of disease respectively. It has been shown that HPV positive disease results in higher survival rates – given many statistics are grouped with oropharyngeal cancer, with a higher incidence of HPV positive cancers, drawing conclusions on survival for oral cavity cancer alone can be difficult. Tumor recurrence is common with oral squamous cell carcinoma, either at the primary site, in the lymph nodes, or as distant metastases in the lungs, liver, or bone.[12] Recurrence is associated with very high mortality and early recurrence is linked with the worst prognosis.

Complications

Complications occur either due to untreated disease and subsequent spread of the tumor or commonly due to side-effects of treatment. Surgery involving tumor excision, neck dissection, and free flap reconstruction carries the risk of flap failure, wound dehiscence, damage to local motor and sensory nerves, vocal cord palsy, trismus, dysarthria, and the potential long-term requirement of tracheostomy and/or feeding tubes. Patients may require an extended stay in intensive care. 

Chemo or radiotherapy can result in a wide range of debilitating, chronic symptoms. Specifically, in the oral cavity, patients may experience mucositis (inflammation of the mucosa) pain, bleeding, trismus, and dry mouth. Together with dysphagia, this can lead to reduced oral intake and malnutrition. Speech can commonly be affected, requiring therapy from Speech and Language teams. Systemic effects of therapy may also result in neutropenia and subsequent associated infections due to immunocompromise. 

The psychological impact of a cancer diagnosis, together with the above complications and side-effects of treatment, can have a devastating and life-long impact on mental wellbeing and quality of life.

reference link: https://www.ncbi.nlm.nih.gov/books/NBK565867/


More information: Marco Fraga et al, Immunomodulation of T Helper Cells by Tumor Microenvironment in Oral Cancer Is Associated With CCR8 Expression and Rapid Membrane Vitamin D Signaling Pathway, Frontiers in Immunology (2021). DOI: 10.3389/fimmu.2021.643298

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