The experimental protocols and the use of human tissues were reviewed and approved by the ethics committees of the National Center for Laboratory Animal Breeding, the Center for Genetic Engineering and Biotechnology, and Hermanos Ameijeiras Hospital (Havana, Cuba). The subjects provided written informed consent for the investigational use of their surgically excised material. These included healthy tissue (dermis and epidermis) serving for control groups derived from healthy female donors undergoing abdominal and facial cosmetic surgery. Malignant samples used in the study consisted of: (1) - Three triple-negative mammary invasive ductal carcinomas (IDCs), which resulted in a high histological grade and intense mitotic index with lymphatic/vascular permeation and confirmed the invasion of sentinel lymph nodes. Donors were white females of ages 34–46 years. (2) - A histologically well-differentiated pancreatic ductal adenocarcinoma tissue was obtained from a 56-year-old white male. (3) - A metastatic melanoma tissue in a cervical lymphatic ganglion was obtained from a 32-year-old black female patient. All the samples were collected during the surgical intervention, washed with ice-cold sterile normal saline to remove fibrin and debris, and cryopreserved in liquid nitrogen until processing for CFF preparation. Tumor sample fragments were fixed in 10% buffered formalin and paraffin-processed for histological analysis, as per protocol. The oncologic samples were ultimately processed and used for the experiments, having received pathology reports of malignancy.

The collected tissue was allowed to thaw and weighed, and approximately 100 mg of wet tissue was placed in a 2-mL vial containing 1 mL of normal saline, which was homogenized using a TissueLyser II for 3 min at 30 revolutions per second. The samples were then centrifuged at 10,000 rpm for 10 min at 4°C, sterilized by filtration using 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 tumor samples were pooled to ensure larger material availability. For the three study protocols described here, protein concentration was used as the arbitrary unit of measurement to prepare and administer the inoculums.

All the biochemical parameters were determined by spectrophotometric methods using commercial kits. Pro-inflammatory markers included C-reactive protein (CRP) [C-Reactive Protein (PTX1) Human ELISA Kit, Abcam, Cambridge, United Kingdom], interleukin (IL)-1β (IL-1 beta Human ELISA Kit, Abcam, Mass, United States), IL-6 (IL-6 Human ELISA Kit, Abcam), and tumor necrosis factor α (TNFα) (Human TNF alpha ELISA Kit, Abcam). Oxidative stress markers included malondialdehyde (MDA) [Lipid Peroxidation (MDA) Assay Kit, Abcam] and H₂O₂ (Hydrogen Peroxide Assay Kit, Abcam). Additionally, Sirtuin-1 (SIRT1) levels were determined using the ELISA Kit for SIRT1 (Cloud-Clone Corp., Houston, Texas, United States). In all cases, the manufacturer’s instructions were followed.

The protocols involving the use of rodents described here were conducted with male BALB/c-Foxn1nu/Cenp mice (body weight 19–22 g and age 12 to 14 weeks) obtained from the National Center for Laboratory Animal Breeding (CENPALAB, Havana, Cuba) and maintained in ventilated racks (Tecniplast, Varese, Italy) in certified rooms for nude mice. Autoclaved food EAO 1004 (CENPALAB, Havana, Cuba) and water were provided ad libitum. Room temperature (20°C–23°C), humidity (65% ± 10%), and the photoperiod cycles (12 h per day) were automatically controlled. The animals were observed twice a day by an experienced staff for monitoring their health status. Body weight was registered a day before study commencement, on a weekly basis, and before the autopsy.

Wild-type adult zebrafish (Danio rerio) were maintained in a recirculating aquatic system facility at the Drug Research and Development Center (CIDEM) under controlled environmental conditions, including a 14-h light/10-h dark cycle, water temperature of 28°C, and pH 7.2–7.5. The fish were fed commercial zebrafish food twice daily, and the system water quality parameters were regularly monitored to ensure optimal breeding conditions and embryo development according to the internal operational procedures.

All the animal procedures were performed according to local and International Guiding Principles for Biomedical Research. All animal studies were conducted under a protocol approved by the Institutional Animal Care and Use Committees of the National Center for Laboratory Animal Breeding (CENPALAB), the Center for Genetic Engineering and Biotechnology, and CIDEM.

The animals were euthanized under terminal anesthesia at the end of the administration period. Alternatively, mice that evolved to an extreme clinical condition during the administration period were sacrificed to ensure a proper autopsy and organ collection (as described below). The autopsy study was conducted following an internal protocol based on described techniques (Scudamore et al., 2014). Gross noticeable changes in organs and tissues were recorded, and fragments were collected for histopathological analysis. Representative fragments from apparently healthy organs were also harvested. Samples were fixed in 10% buffered formalin and paraffin-embedded, and serial 5-μm sections were stained using H&E. Images were captured using a BX53 Olympus microscope coupled to a digital camera and central command unit (Olympus Dp-21). Histological examinations were blindly performed by three MD pathologists experienced in experimental pathology (EAH, DDC, and LLM). The histopathological findings of premalignant and malignant lesions were collectively discussed and ultimately diagnosed in accordance to the current recommendations (Nikitin et al., 2004; NTP, 2023). Paraffin sections of representative tissue lesions in mice receiving the tumor material and the corresponding specimens from control counterparts were mounted on poly-l-lysine-coated slides (Dako, California, United States) in order to reduce inter-tissue/experimental variations during immunohistochemistry studies. The slides were dewaxed and rehydrated through graded washes with ethanol. The rehydrated slides were exposed to a high-pH antigen retrieval solution (Dako, United States) for 20 min at 90°C. Following equilibration at room temperature, the slides were washed in PBS and blocked with endogenous peroxidase. Unspecific binding blocking solution was used for 20 min, and the sections were incubated for 40 min with antibodies directed to TTF-1 (1/100; Santa Cruz Biotechnology SC-53136), c-Myc (1/100; Abcam, ab32072), proliferating cell nuclear antigen (PCNA) (1/250; Cell Signaling Technology (PC10) Mouse mAb #2586), estrogen receptor (1/250; Santa Cruz Biotechnology (D-12) SC-8005), progesterone receptor (1/250; Santa Cruz Biotechnology, FKBP51 SC-271547), HER-2 (1/200; Santa Cruz Biotechnology, ErbB2/HER2 (A-2) SC-393712), telomerase reverse transcriptase (TERT) (1/150; Abcam, ab216625), survivin (1.200; Abcam, ab469), CD56 (1/200; Invitrogen, Cat #PA5-78402), CD45 antigen (leukocyte common antigen, 1/100; Abcam, ab10558), and cytokeratin 7 (CK7) (1/250; Abcam, ab181598). Antigens were retrieved according to each antibody manufacturer’s instructions and protocol. The immunolabeling reaction was developed as described for the Mouse- and Rabbit-Specific HRP/DAB (ABC) Detection IHC kit (Abcam, ab64264). Non-specific tissue labeling internal controls included the omission/replacement of the primary antibody by the background-reducing antibody diluent and normal rabbit serum (Boster Biological Technology, Pleasanton, CA, United States, catalog # AR1010).

After 60 h of incubation with the pathologic and healthy tissue-derived homogenates and the other controls, the embryos were processed for immunocytochemistry, as previously described (Guille, 2008). In brief, embryos were rinsed with PBS and fixed in 4% paraformaldehyde at 4°C for 1 h and post-fixed in acetone at −20°C on poly-l-lysine-coated slides. Before incubation with the primary antibody, the embryos were soaked in a background-reducing protein block (Abcam 156024) for 15 min at room temperature. Subsequently, the embryos were incubated with anti-c-Myc (1/250) and anti-HER-2 (1/500) antibodies for 20 min at room temperature. After three washes with PBS, the slides were incubated with a biotinylated secondary linker and washed with horseradish peroxidase (HRP)-labeled streptavidin solution, as previously described (Abcam, 2024). Primary antibody binding is identified by the reddish-brown stain due to the HRP reaction with diaminobenzidine. The reaction development was stopped by PBS washes, and the coverslips were mounted with ClearMount™ Mounting Solution (Invitrogen). No contrast was used. Immunolabeled cells were identified through anatomic illustrative descriptions of zebrafish embryos (von Hellfeld et al., 2020).

Statistical analyses were performed using GraphPad Prism 10.1.0 software. Normal distribution was analyzed using D’Agostino–Pearson and Shapiro–Wilk normality tests. Variance homogeneity was evaluated using Brown–Forsythe and Bartlett’s tests. The mice groups were compared during the study using two-way ANOVA, followed by Tukey’s multiple comparisons test. Body weight changes were processed, and inter-groups were compared through the mixed-effects model (REML) method and Dunnett’s multiple comparisons test. Survival data were analyzed using the log-rank Mantel–Cox test. p-values < 0.05 indicated statistically significant differences.

As described, surgical pieces of healthy donor skin and fragments of three different human malignant tumors were used to prepare the CFFs. The elemental biochemical description shown in Table 1 indicates that cellular lipids undergoing a peroxidation process are similar among the healthy skin and the tumor samples, as reflected by the MDA levels. However, metastatic melanoma generates superoxide anion and hydroxyl radicals, as judged by the H₂O₂ levels, which exceeded the values determined for the healthy control skin and other malignant samples by between 4 and 6-fold. Tumor-derived samples seemed to exhibit a particular inflammatory signature. CRP appeared particularly elevated in pancreatic adenocarcinoma cells, exceeding the concentration detected in metastatic melanoma and healthy control skin by over 24-fold. Melanoma-derived CFFs, however, showed the highest level of IL-6, followed by pancreatic adenocarcinoma cells. In line with this, melanoma also exhibited the largest concentration of IL-1β, TNFα, and Sirtuin-1, which was followed by the concentration determined in mammary carcinoma homogenates. Of note, healthy donor skin tissue homogenates showed the lowest values of IL-1β, while IL-6, TNFα, and Sirtuin-1 were not detected (Table 1).

The administration of mammary ductal carcinoma CFFs was not associated with behavioral changes, clinical deterioration, or severe/acute somatic emaciation. As shown in Figure 1, the effect of the mammary carcinoma homogenate significantly impacted the animal body weight from the fifth week onward compared to the recipients of the healthy donor homogenates. This mammary cancer CFF did not affect animal survival during the effective administration period of 8 weeks.

The administration of breast carcinoma homogenates generated premalignant and malignant changes in lung tissue. Microscopic examination of the lungs in all these mice (N = 8) exhibited extensive areas of parenchymal condensation, in which atypical adenomatous hyperplasia (AAH) predominated at the expenses of type 2 pneumocytes with clear, enlarged, and atypical nuclei (not shown). In these areas of parenchymal condensation and alveolar luminal encroachment, large subpleural nodules diagnosed as adenocarcinoma of both solid and lepidic growth were distinguished (Figure 2A). Mice treated with the healthy skin homogenate showed morphologically normal pulmonary parenchyma (Figure 2B). The immunohistochemical characterization of these nodules confirmed their authentic malignant nature. Tumor cells exhibited a strong nuclear signal for TTF-1 (Figure 2C), which contrasted with the restricted expression of the control specimens (Figure 2D). These cells also exhibited remarkable cytoplasmic HER-2 expression (Figure 2E) compared to controls (Figure 2F).

Broad nuclear expression of PCNA was observed in the tumor-bearing mice (Figure 2G) compared to the expression in the lung normal parenchyma of healthy skin-treated mice (Figure 2H). Nuclear and cytoplasmic c-Myc overexpression that extended to areas of microscopically normal alveolar walls was found in the lungs of breast-tumor inoculated mice (Figure 2I). Control mice alveolar walls, however, exhibited only marginal immunolabeling (Figure 2J). Meaningful nuclear TERT expression was observed in these tumors, concurrent with the overexpression of nuclear survivin, nuclear estrogen, and progesterone receptors as part of the tumor phenotypes (not shown). Within these tumors, conglomerates of cells appeared organized, forming rosette-like structures that encircled the arterioles and bronchioles. The rosette-like aggregates were heterogeneous and contained large, atypical, irregular, epithelial, CK7-positive cells (not shown) and small, round, blue cells that often appeared with large-sized vesiculous nuclei with lax chromatin and central conspicuous nucleoli. Immunohistochemistry demonstrated that some of these cells were negative for CD45 (not shown), whereas some cells were found positive for CD56, suggesting its neuroendocrine origin (Figure 2K). These round blue cells were often visualized forming subpleural, periarteriolar, and peribronchial nodules with notorious nuclear molding, presumptively identified as small-cell lung carcinoma (Figure 2L).

Finally, an interesting immunohistochemical finding was the presence of HER-2, CK7, estrogen, and progesterone receptor-positive cells in the subcapsular sinus of thoracic lymphoid nodes. Intense nuclear labeling for both hormone receptors was observed in these cells. Figure 2M shows the intense expression of the progesterone receptor; lymphatic node samples from the mice control group were negative for HER-2 and CK7. Faint staining for progesterone (Figure 2N) and estrogen receptors appeared circumscribed to the cell membrane.

After 6 injections of the tumor CFF, animals began to show asthenia, a tendency of isolation, hunched posture, and frail, dry, and flaky skin. Although the dose was de-escalated to 50 µg in the second week in order to prevent animal decay, the aspect of “ill condition” continued with evident emaciation. Severe and progressive cachexia debuted in the third week. A significant body weight loss was observed from that point onward compared to healthy control recipients (Figure 1). Four animals with ostensible clinical deterioration were untimely euthanized and autopsied, whereas the other four mice, although in a cachectic state, completed 8 weeks of administration. Healthy donor skin-treated mice remained active and healthy during the administration period.

The histopathologic examination of mice organs exposed to pancreatic tumor CFFs revealed that, again, the lungs appeared to be the major target structure with pre- and malignant changes. Two major findings were obtained: multiple foci of solid, poorly differentiated adenocarcinoma across the lung parenchyma tissue (Figure 3A) and marked hyperplasia of pancreatic Langerhans islets (Figure 3B). Alveolar/bronchiolar hyperplasia and atypical adenomatous hyperplasia (AAH) were the only findings attributable to the pancreatic tumor CFF in mice that were prematurely autopsied. Nevertheless, immunohistochemistry revealed a strong nuclear and cytoplasmic TTF-1 expression in the bronchoalveolar cells within these foci of AAH (not shown). None of the mice treated with the healthy donor skin homogenates exhibited microscopic pathologic changes, including the normal aspect of Langerhans islets (Figure 3C).

Closely resembling the effect of the pancreatic tumor homogenate, the administration of 50 µg of melanoma-derived protein also led to a cachectic process and clinical involution from the third week of treatment onward (Figure 1). Consequently, the mice were untimely and gradually autopsied on days 34, 36, 43, 45, 49, and 55 due to their morbid clinical aspect. On day 61, the only two remaining mice were autopsied, and the administration period was concluded for this protocol. The mice treated with the healthy donor skin remained normal and active during the 2-month administration period. Two mice of this group were found dead on days 57 and 60 with evidence of autolysis.

Melanoma-derived CFF administration, in addition to exerting the described cytotoxic effect, induced a broad spectrum of pre-malignant and malignant proliferative changes that involved the lungs, liver, kidneys, and skin. Bronchoalveolar proliferative changes extended from AAH to intra-alveolar pseudopapillary adenocarcinomas, which were made up of round blue cells with atypical and overlapping irregular nuclei, exhibiting hyperchromasia and clumped chromatin. Another cell population observed in this conglomerate showed large vesiculous nuclei with a coarse dispersed chromatin and prominent nucleoli (Figure 4A). In line with this pathologic proliferation in the lungs is the identification of circulating tumor cells as an embolus within a lymphatic vessel lumen, integrated by a cluster of cells with the same cytological traits as described above (Figure 4B). In relation to the liver, two major alterations were identified (Bordonaro, 2019): the presence of nodules or aggregates of cells of putative leukocytic origin with small, irregular, and atypical nuclei scattered across the liver parenchyma (not shown) (NCI, 2021) and the presence of voluminous hepatocytes with large, atypical, and variegated nuclei containing a coarsely granular or dispersed chromatin (Figure 4C). Normal liver histology was observed in the group of control mice (Figure 4D). In the kidney parenchyma, infiltration of neoplastic cells was found in the interstitium, peritubular capillaries, and the lumen of the tubules, demonstrating cellular atypia, irregular nuclei, pleomorphism, and prominent nucleoli. An interstitial inflammatory infiltrate of lymphocytes and polymorphonuclear neutrophils with a desmoplastic reaction was also observed (Figure 4E). On the contrary, animals from the control group showed no pathological changes in kidney parenchyma (Figure 4F). Skin of melanoma-treated mice also revealed an abnormal and irregular epidermal proliferative disorder, which included the epidermis and epithelial dermal ridges. Epidermal cells showed notable nuclear overlapping, the presence of irregular, sharply angulated, and hyperchromatic nuclei, mitotic figures, and prominent nuclear atypia (Figure 4G). The skin of the control mice showed no histopathological alterations (Figure 4H).

The incubation of zebrafish embryos with 100 μg/mL of protein from breast ductal carcinoma homogenates for approximately 60 h proved to elicit the expression of c-Myc and HER-2 in stark contrast to embryos exposed to healthy donor skin (Figure 5). A constellation of embryo cells was found to express both tumor markers. These included melanocytes, notochord, cephalic, and eye cells. Of note, incubation with malignant tissue homogenates acutely altered the embryo homeostasis. The number of heartbeats/minute was significantly reduced at the 24-h time point and during the two subsequent evaluation points. Other morphological non-lethal anomalies, such as hypopigmentation and spine curvature, were observed at 48 and 66 h of evaluation. Exposure of embryos to healthy donor-derived CFFs (control of human material) did not induce functional or morphological changes in the embryos, similar to the two other controls included (Table 2).