Induction of Premalignant and Malignant Changes in Nude Mice by Human Tumors-Derived Cell-Free Filtrates

Research Article

Ann Hematol Oncol. 2022; 9(1): 1387.

Induction of Premalignant and Malignant Changes in Nude Mice by Human Tumors-Derived Cell-Free Filtrates

Berlanga-Acosta J¹*, Mendoza-Mari Y¹, Martinez-Jimenez I¹, Suarez-Alba J¹, Rodriguez-Rodríguez N¹, Garcia-Ojalvo A¹, Fernandez-Mayola M¹, Rosales-Torres P², Arteaga-Hernandez E³, Duvergel-Calderin D³, Borrajero-Martinez I³, Campal-Espinosa A¹, Fuentes-Morales D4, Pimentel-Vazquez E¹, Zamora-Sanchez J¹, Martinez-Suarez JE³, Estevez-del Toro M³, Gomez-Cabrera E5 and Guillén-Nieto G¹

1Center for Genetic Engineering and Biotechnology, Biomedical Research Direction, Ave 31 SN e/158 and 190, Cubanacan, Playa 10600, Havana, Cuba

2Department of Pathology, Maria Curie Oncology Hospital, Carretera Central Oeste esquina Madame Curie, Camagüey, CP, 70 700, Cuba

3Department of Pathology, Hermanos Ameijeiras Hospital, Calle San Lazaro # 701, Centro Habana, La Habana 10400, Cuba

4National Center for Laboratory Animal Breeding, Calle 3ra, No 40759, Entre 6ta y Carretera de Tirabeque, Reparto La Unión, Boyeros, La Habana, Cuba

5National Institute of Oncology and Radiobiology, Calle 29 esquina a F, CP 10 400, Vedado, La Habana, Cuba

*Corresponding author: Jorge Berlanga-Acosta, Center for Genetic Engineering and Biotechnology, Ave, 31 S/N, e/158 and 190, Cubanacan, Playa 10600, Havana, Cuba

Received: December 20, 2021; Accepted: January 24, 2022; Published: January 31, 2022


Carcinogenesis is a vast and heterogeneous, multi-step process driven by genetic and epigenetic operators leading to superimposed “tumor organs”. Based on our previous experiences of reproducing human donor’s histopathological hallmarks in healthy animals exposed to pathologic tissues homogenates, we examined the consequences of administering crude homogenates elaborated from invasive human tumors to nude mice. Subcutaneous administrations of increasing protein concentrations of breast carcinoma homogenates for 6 and 12 weeks, induced lungs atypical adenomatous hyperplasia, foci of lepidic and solid growth poorly-differentiated adenocarcinomas at the two time points. Non-atypical mucosal hyperplasia and adenomas were detected along the gastrointestinal tract. Another experiment addressed the impact of daily administrations (100 μg protein/mouse) of anaplastic sarcoma tissue homogenate for a month. This scheme triggered proliferative changes including: lung adenocarcinomas; a subcutaneous, poorly-differentiated mesenchymal cells tumor, a lymphoadenoma, and multiple gastrointestinal adenomas. When 50% of the month-treated animals were left to evolve treatment-free for other 35 days, a larger and broader incidence of neoplastic changes was found, suggesting autonomous growth: lung adenocarcinomas, a poorly differentiated thyroid tumor, an epithelial tumor within the periaortic brown adipose tissue, and multiple adenomas. These findings indicate that tumor crude homogenates contains soluble “transforming” messengers, that in a short period of time disrupt tissues’ proliferative and differentiation programs drifting to progressive neoplasms. This study expands previous evidences on the ability of human pathologic tissues-derived homogenates, to induce the reproduction of diseased donor’s histopathologic hallmarks.

Keywords: Cancer; Breast tumors; Sarcoma; Malignant transformation; In vivo carcinogenesis; Nude mice; Cells-free filtrate


CFF: Cells-Free Filtrate (s); IDC: Invasive Ductal Carcinoma (s); CRP: C-Reactive Protein; IL-6: Interleukin-6; TNF-a: Tumor Necrosis Factor-a; VEGF: Vascular Endothelial Growth Factor; MDA: Malondialdehyde; ΔΔCt: Ct Method; RPLE13A: Ribosomal Protein L13a; YWAHZ: Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta; CEA: Carcinoembryonic Antigen; TTF-1: Thyroid Transcription Factor-1; PPAR-γ: Peroxisome Proliferator-Activated Receptor Gamma; EGFR: Epidermal Growth Factor Receptor; c-Myc: Multifunctional Transcription Factor; PCNA: Proliferating Cell Nuclear Antigen; TGF-a: Transforming Growth Factor Alpha; VEGFR 2: Vascular Endothelial Growth Factor Receptor 2; WBC: White Blood Cell; RBC; Red Blood Cell; LDH: Lactate Dehydrogenase; ALAT: Alanine Aminotransferase; ASAT: Aspartate Aminotransferase; TP53: Tumor Suppressor Protein p53; RB1: Retinoblastoma Protein; EGFR: Epidermal Growth Factor Receptor; CCND1: Cyclin D1 Coding Gene; HIF1A: Hypoxia Inducible Factor 1 Subunit Alpha; BRCA1: Breast Cancer 1; ERBB2: Ligand Orphan Receptor; Member of the Epidermal Growth Factor Receptor Family; AAH: Atypical Adenomatous Hyperplasia; BAT: Brown Adipose Tissue


Cancer research dates back to 2,700 years ago [1], and it is likely that the biological foundations of no other human pathology has been so extensively and comprehensively investigated. Cancer is in fact a heterogeneous group of diseases that has remained for years as a major cause of worldwide mortality [2].

The process of cellular malignant transformation entails a sequence of stochastic and non-stochastic events [3] that has enticed, baffled, and discouraged researchers for centuries. By no other means could it be, since carcinogenesis is an enormous-in-size process, that stems from variegated genetic alterations including loss and gain of function mutations that can occur in one single cell [4,5], which runs along stepwise changes from initiation to promotion and progression [6,7], and that translate in enormous cellular resilience for survival, proliferative and dissemination capabilities [8,9]. Cancer cells are also endowed with the singular marks of immortality and autonomy even in adverse tissue ecosystems [1,10]. These cells stripes are able to dedifferentiate [11,12] disguising from their original phenotype, so as to implement transitional reprogramming events from epithelium to mesenchyme [13]. For years, cancer has bewildered pathologists given the hallmarks of atypia, pleomorphism and heterogeneity 14,15] which are amplified by consecutive adaptations to micro environmental pressures [16,17]. Cancer cells are also gifted with the unusual faculty to locally and remotely invade, colonize, adapt and live in distant tissues, even when the embryonic origin of the metastatic niche may not match with that of primary tumor cells. Ultimately, metastasizing cells enslave the new substrate [18-21]. This multi-faceted process of metastatic seeding has an extremely low mathematical probability (10-8), nonetheless the majority of deaths from solid tumors are caused by metastases [22,23]. Malignant cells also educate the host immune system with lessons of tolerance and ironically use inflammation for their own benefit [11,24]. Finally, cancer cells are actual messages-editing plants, being able to produce and deliver a broad variety of encapsulated or free signals with multiple pathological implications [25,26]. These cancer hallmarks [27,28], has made cancer to immovably remain for years as the emperor of maladies [29].

After Bernard Peyrilhe inaugurated the investigative use of human cancer-derived fluids, the administration to animals of filtered cell-free tumor extracts translated in the groundbreaking discovery of leukemia and sarcoma-causing viruses [30]. We recently undertook the use of cells-free filtrates [31] to examine the hypothesis that the chemical codes of diabetes-related archetypical histopathologic hallmarks in nerves and vessels could be passively transferred to healthy animals, and accordingly reproduce these changes in a way mirroring those of the donor tissues. We therefore envisioned that cells-free filtrate (CFF) could be the vehicle to deliver metabolic memory-associated signalers, acting as driving forces to impose microangiopathy and neuropathy in a normal recipient animal. Having successfully reproduced diabetic vascular pathology in rats [31], CFF were elaborated from lower limb atherosclerotic arteries derived from amputated patients affected with chronic limb ischemia. It was reiteratively observed that CFF inoculation to healthy rats, recreated arteriolar walls histopathological changes typically described for this condition, and identified in the donor subjects (submitted-Frontiers in Cardiovascular Medicine). Once again, the filtered-whole tissue homogenate in a physiological saline solution facilitated the transference of some kind of tissue-derived signal that accounted for the reconstruction of the donor pathology in the host animal.

In line with these notions, and considering that carcinogenesis doctrines entail the combined participation of genomic mutations [32] and epigenetic derangements, as determinants for malignant initiation, progression, and ultimately immortality and autonomy [10,33-35], we embarked on experiments in which healthy nude mice were inoculated with fresh human tumor-cells free filtrates, assuming the hypothetical transference of donor’s-derived carcinogenic drivers. We report here that the administration of these tumors crude material is ensued by the onset of progressing premalignant and malignant changes in a narrow temporary window in otherwise normal recipient animals.

Materials and Methods

Ethics and consent

The experimental protocols and the use of human tissues were reviewed and approved by the ethic committees of the National Center for Laboratory Animal Breeding, and Hermanos Ameijeiras Hospital (Havana city, Cuba) respectively. Subjects provided written informed consent for the use of their surgically excised material. These included healthy tissue (dermis and epidermis) serving for control groups, derived from a 42-years old healthy female donor undergoing abdominal cosmetic surgery. Malignant samples used in the study consisted of (1) three mammary invasive ductal carcinomas (IDC) which resulted in high histological grade and intense mitotic index, with lymphatic/vascular permeation, and confirmed invasion of three to five sentinel lymph nodes. (2) A voluminous intrathoracic pleomorphic anaplastic sarcoma that invaded the right hemithorax of a 37-years old female patient. During surgery collected samples were washed with sterile ice-cold normal saline to remove fibrin and debris, and cryopreserved in liquid nitrogen until processing for the CFFs preparation. Tumor samples fragments were as routinely 10% buffered formalin fixed and paraffin processed for histological analysis. The oncologic samples were ultimately processed having received pathologists’ report of malignancy.

CFF preparation

Collected tissue was allowed to thaw, weighed and approximately 100 mg of wet tissue were placed in 2 mL vial containing 1 mL of normal saline, homogenized using a Tissue Lyser II for 3 minutes at 30 revolutions per second. Samples were then centrifuged at 10 000 rpm for 10 minutes at 4°C, sterilized by filtration through 0.2 μm nitrocellulose filters (Sartorius Lab Instruments), aliquoted into sterile Eppendorf vials and stored at -70°C. Given the histological similitude of the IDC samples, the three tumors were pooled to ensure larger material availability. Protein concentration was used as the arbitrary unit of measurement to prepare and administer the inoculums.

Characterization of the CFF

Protein concentration (Bicinchoninic Acid Protein Assay Kit. Sigma-Aldrich, USA), and standard commercial ELISA kits for human C-Reactive Protein (CRP), Interleukin-6 (IL-6), tumor necrosis factor-a (TNF- a), and Vascular endothelial growth factor (VEGF) (All from Abcam, Massachusetts, USA) were used according to manufacturer instructions in order to characterize the different tissue-derived CFF. Malondialdehyde was selected as indicator of lipid peroxidation (Lipid Peroxidation Assay Kit, Abcam).

Nucleic acids determination, gene amplification and expression from the tissues homogenates

Two microliters of each CFF were used to determine the DNA and RNA concentrations using a NanoDrop spectrophotometer. The measurement at 260 nm was correlated with nucleic acids concentrations. Total RNA was isolated from 500 μL of each CFF the RNeasy Lipid Tissue Mini kit (Qiagen, Germany). Briefly, 1 mL of Qiazol reagent was added to each sample, mixed by vortex, treated with 200 μL of chloroform and vigorously shaken during 15 seconds. The mixtures were then centrifuged at 12 000 rpm for 15 minutes, 4°C. Upper aqueous phase was transferred to a clean tube and the same volume of 70% ethanol was added and gently mixed. The samples were completely transferred to RNeasy Mini spin columns and washed with 350 μL of buffer RW1. On-column DNase digestion was performed according to Qiagen standard protocol. Subsequently, RNAs were washed with 350 μL of buffer RW1 and twice with 500 μL of buffer RPE. RNAs were eluted in 30 μL of RNase-free water. Concentration (ng/μL) and quality (260/280 nm ratio) of each RNA were estimated by Nanodrop spectrophotometer. Complementary DNAs were obtained from 50 ng of total RNAs, using Invitrogen SuperScriptTM III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, Carlsbad, USA) kit, following manufacturer instructions. All cDNAs were diluted 1:10. This methodology was similarly and concurrently applied to corresponding solid tissue samples (pathologic and healthy).

Quantitative PCR reactions were set up in 20 μL using LightCycler® 480 SYBR Green I Master 2x (Roche, Germany) and 300 nM mix of oligonucleotides for each gen (Table 1-Supplemental material). The runs were carried out in a LightCycler®480II (Roche, Germany) and three technical replicas per sample were performed. A standard SYBR Green Probe II program with 45 cycles was used. Reference genes for normalization were chosen according to the stability index after geNorm analysis [36]. Efficiency value for each oligonucleotide pair was calculated by LinRegPCR software (version 11.3) [37]. Fold change for each gen and sample was calculated by REST 2009 [38], using Ct and efficiency values. This software uses the comparative Ct method (ΔΔCt) to analyze data. Gene expression levels were normalized with endogenous control genes YWHAZ (tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein zeta) and RPLE13A (ribosomal protein L13a). For expression levels >1, fold change was considered the same; for expression levels between 0-1, fold change was expressed as -1/expression value. Expression data statistical analysis was performed by REST 2009 software, which uses pair-wise fixed reallocation randomization test. Statistical significance was established for p-values lower than 0.05.