Oncosuppressor-Mutated Cell-Based Diagnostic Platform for Liquid Biopsy Diagnoses Benign Head and Neck Masses and Predicts Malignancy in Thyroid Nodules: Results from a Consecutive Cohort of Patients

Abstract

<b><i>Background:</i></b> We reported that a novel oncosuppressor-mutated cell (OMC)-based platform has the potential for early cancer detection in healthy individuals and for identification of cancer patients at risk of developing metachronous metastases. <b><i>Objective:</i></b> Herein, we sought to determine the diagnostic accuracy of this novel OMC-based platform in a consecutive cohort of patients operated for suspicious head and neck masses. <b><i>Methods:</i></b> OMCs (<i>BRCA1</i>-deficient fibroblasts) were exposed to blood serum from patients with head and neck nodules before surgical removal. These cells were analyzed for their proliferation and survival. Treated OMCs were inoculated subcutaneously in NOD/SCID mice, and tumor growth was monitored over time. <b><i>Results:</i></b> OMCs exposed to serum from patients with malignant lesions displayed increased proliferation compared to those exposed to serum from patients with benign lesions. Only OMCs exposed to serum from patients diagnosed with malignant thyroid neoplasia generated a cancerous mass. The sensitivity of the test was 92%, with only 1 false negative out of 34 patients. Immunohistochemical staining showed that the cancerous masses were poorly differentiated adenocarcinomas with high proliferative index. <b><i>Conclusions:</i></b> These data show that liquid biopsy combined with an OMC-based in vivo platform has the potential to diagnose benign head and neck masses and predict whether a thyroid nodule is malignant. These results strengthen the concept that OMCs can be used to detect circulating malignant factors in cancer patients.

Horizontal Transfer of Malignant Traits and the Involvement of Extracellular Vesicles in Metastasis

“…The HTMT model concurs with the genometastatic hypothesis that the metastatic process is not due to the migration of cancer cells per se through the general circulation, as postulated in the Seed and Soil model, but to the transfer of genetic material. The novelty of HTMT is based on the following five observations and postulates: (A) oncogenic information is carried by small EVs circulating in the blood of patients even at the precancerous stages, i.e., when tumor cells have not yet reached the stage of full malignant transformation [ 179 , 181 , 182 ]; (B) the mutation of at least one oncosuppressor gene in recipient cells in the PMN induces the expression of proteins that facilitate the uptake of cancer EVs [ 70 , 180 ]; (C) the phenotypic changes induced in recipient cells depend on the type of cell that uptakes the EVs rather than the source of the oncogenic message [ 179 , 183 ]; (D) the ability to evade the immune system can be transferred from the primary cancer to distant cells through cancer EVs [ 153 , 179 ]; and (E) metachronous metastases could be due to the nuclear integration of key EV-derived cancer-related genes that are expressed after a period of latency [ 153 ].…”

“…The discovery that, according to the HTMT model, EVs circulating in patients with neoplastic disorders, ranging from dysplastic to metastatic lesions, have a transforming ability on oncosuppressor-KO cells (see below), is the basis of a cancer screening test that can distinguish healthy patients from patients with cancer or at risk of developing cancer [ 181 , 182 , 183 ]. This ability displayed by oncosuppressor-mutated cells to be transformed by cancer EVs has been incorporated in a unique biological platform for cancer screening called MATERD ( M etastatic A nd T ransforming E lements R eleased D iscovery platform) [ 181 , 182 , 183 ]. Among the peculiarities of this system is its intrinsic ability to detect neoplastic disorders even in patients who had only dysplastic lesions or cancers in situ, and its ability to detect cancer factors even years after the resection of the primary tumor to predict metastatic recurrence.…”

“…In this regard, MATERD sensitivity is higher than the latest liquid biopsy tests based on the detection of circulating cancer cells, circulating DNA and circulating miRNA [ 184 , 185 , 186 ]. The clinical results obtained with the MATERD test confirm that (i) cancer factors circulate in the blood prior to the complete cancerous transformation of the cells, undermining the concept that cell circulation is paramount to metastasis formation in distant organs [ 181 , 182 , 183 ], and (ii) cancer factors are still found circulating in the serum after primary cancer has been removed and for several years afterwards, therefore strengthening the concept that these factors do not require the presence of cancer cells to induce metastatic disease [ 181 , 182 , 183 ].…”

“…For example, when immortalized cell lines such as human HEK293 kidney embryonic cells, PNT-2 prostate cells [ 188 ], which exhibit alterations in cell cycle control, MCF10 cells carrying a mutated oncosuppressor gene, like phosphate and tensin homolog ( PTEN ), or the human fibroblast cell line carrying a CRISPR-Cas9-based deletion of the tumor suppressor breast cancer gene 1 ( BRCA1 ) are exposed to blood serum of cancer patients or EVs derived therefrom, they transform into malignant cells which, upon subcutaneous injection into immunosuppressed mice, give rise to cancer masses [ 178 , 180 , 181 , 182 , 183 ] ( Figure 2 ). In contrast, unaltered cells such as human embryonic stem cells, adult liver fibroblasts and MSCs do not [ 178 ].…”

Cellular Adaptation Takes Advantage of Atavistic Regression Programs during Carcinogenesis

“…In both cases, microenvironmentinduced changes should not be underestimated [114], since the TME is a milieu where the cancer-associated network drives metabolic reprogramming, leading to cancer onset and progression, as well as horizontal transfer of neoplastic traits, but also to the development of therapeutic resistance due to the release of extracellular vehicles (EVs) [115]. Indeed, recently, it has been described that EVs delivered by human cancer cells (known to contain genetic material and signaling molecules) may be up-taken by onco-suppressor-mutated cells (OMCs), which become malignant with the phenotype compatible with those of the cancer cells from which the EVs were derived [116]. In addition, EV release in cancer has also been described as an adaptation to a microenvironmental selection pressure that further promotes the survival of cancer [117]; thus, the release of EVs may occur in (or be controlled by) specific microenvironmental conditions before circulating oncogenic factors are present in circulation.…”