New Evidences about Unusual Behavior of HeLa Cells in Stress Environment Concerning Immortality Status

1.1 Characteristics of papillomaviruses

The term “papillomavirus” originated from the Latin for nipple (papillo) and tumor (ome). Papillomaviruses (PVs) are nonenveloped DNA viruses that induce exophytic lesions of the skin and mucosal surfaces. PVs have been around for millions of years; fossil remains of ancient organisms have been found to contain PV DNA.

About 450 genotypes of PVs have been documented, with 12 types characterized as carcinogenic [1]. PVs have been found in all living vertebrates and invertebrates, including birds, turtles, snakes, shellfish, and so on.

The first animal PV was described in 1933 by Richard Shope who investigated papillomata in “warty” wild cottontail rabbits, also known as “jackalopes” [2]. These animals were infected with PVs and formed benign tumors on the head as horns and on the body together with keratin plaques. But if this benign tumor was minced and infected to health cottontail rabbit, there were formed carcinogenic tumors then on the body.

In 1975, Harald zur Hausen published the hypothesis that human papillomavirus (HPV) played a specific inductive role in the etiology of cervical cancer. For this work, he was awarded a Nobel Prize in Physiology and Medicine in 2008 [3].

Each HPV genome contains six structural genes within the “early” region: E1, which encodes helicase; E2, known as a suppressor of the expression of oncogenes E6 and E7; and E4 and E5, with functions that are not fully understood. Additionally, there are two genes in the “late” region, which encode the major L1 coat protein and the minor L2 coat protein.

HPV types 16 and 18 are responsible for approximately 70% of cervical cancers and precancerous cervical lesions. It is noteworthy that up to 50 genomic copies of HPV18, which have undergone recombination with cellular sequences, can be identified in HeLa cells. These cells, derived from the cervical carcinoma tissue of a 31-year-old patient, were the first human cells to be successfully cultured in vitro. HeLa cells have since become widely used as a laboratory cell line [4].

Comparing the behavior of immortal HeLa cells with that of non-immortal bacteria surviving in biofilms could offer valuable insights into cellular immortality and microbial survival strategies.

1.2 The conditions of biofilm formation in connection with HeLa cells

Biofilms are complex communities in which resident bacteria exist within a self-derived extracellular matrix. Every bacterial species, or consortium thereof, forms its own unique multicellular community [5]. Architectural and behavioral investigations of biofilms of different bacteria have led to the conclusion that biofilm formation on both living and inanimate surfaces serves as an adaptation for survival, allowing bacteria to overcome environmental threats such as antimicrobial agents and host defense mechanisms. However, as biofilms mature, they encounter challenges such as nutrient limitation and accumulation of waste products. As such, biofilm-associated bacteria may return to a planktonic state, which offers advantages in nutrient availability [6]. To facilitate the study of the association between biofilm formation and the virulence determinants of pathogenicity, different ex vivo models have been developed. These models offer more strictly controlled experimental conditions compared to in vivo models, allowing for intensive research. Ex vivo models are also useful for assessing therapeutic pathways for combating biofilm-related infections [5].

Nevertheless, biofilms have had a global impact on modern medical and microbial literature, but their study continues to raise more questions. Despite extensive research, there is lack of evidence regarding the formation of biofilms by HeLa cells.

In a search spanning 5 years, only a few instances were found suggesting the involvement of HeLa cells in biofilm formation with various bacteria species. Examples include enteropathogenic and uropathogenic Escherichia coli [7], Providencia stuartii [8], Candida tropicalis [9], Aliarcobacter butzleri [10], mastitopathogenic E. coli [11], and Lactobacilli [12]. However, these instances typically involve bacteria settling on certain areas of HeLa cell surfaces without forming cohesive structures or consortia. It appears that HeLa cells remain largely unaffected by these bacterial interactions.

The limited evidence suggests that HeLa cells may possess resistance to invasion by other bacteria. This could imply that the cell walls and membranes of HeLa cells are highly stable and able to withstand the presence of other organisms.

Nevertheless, certain natural products can destroy HeLa cells. For example, extracts from medicinal cactus plant Morinda citrifolia [13] were found to induce aging and loss of adhesion of HeLa cells, leading to their detachment and as single cells. This suggests that HeLa cells possess an integral surface membrane that can be affected by certain compounds. Similarly, the antimicrobial peptide brevedin, derived from the skin of lake frogs, has been shown to permeate bacterial membranes via endocytosis and inhibit the proliferation of HeLa cells [14]. Furthermore, a cytotoxic extract from the anticancer medicinal plant Vassobia breviflora [15] has been found to block the growth of HeLa cells at a concentration of 0.03 mg/ml. This extract also exhibits antibiofilm activity against other bacterial species.

It was of interest to investigate the potential anti-HeLa cell activities of well-known oncolytics such as HPV16 E2, L-amino acid oxidase (LAAO), and D-amino acid oxidase (DAAO). The literature has documented the apoptotic activity [16, 17, 18, 19, 20, 21, 22, 23, 24, 25] of HPV16 E2, its antiproliferative properties [24], and anticancer action [26, 27] alongside the well-documented activities of LAAO [27, 28, 29] and DAAO [6, 27, 29].

Therefore, the goal of this study is to investigate the effects of superoncosupressor “early” protein HPV16 E2, LAAO, DAAO, and other “early” proteins like HPV16 E6 and HPV16 E7 on HeLa cells ex vivo. The study aims to elucidate whether these proteins could potentially “educate mice” splenocytes to recognize or learn to target lung tumor cells induced by HeLa cells, ultimately driving them into regression. This approach holds promise for developing novel therapeutic strategies against HeLa cell-derived tumors by harnessing the immune system’s ability to recognize and eliminate cancerous cells.

3.1 The action of HPV16 Е2, vaccine material of tomato transgenic with “early” genes HPV16 Е2, Е6 and Е7, therapeutic proteins LAAO and DAAO on the cultivation of cancer HeLa cells in vitro

Before conducting the experiments, the suspension of HeLa cells was kept in a low-temperature freezer in DMEM medium supplemented with 30% glycerol or DMSO. During seeding, 0.5–1 ml of unfrozen suspension of HeLa cells was placed in 5 ml of DMEM medium with the addition of 10% of fetal cow serum and antibiotics penicillin 500 units and streptomycin 50 mg/l. After 30 minutes of seeding, a monolayer of HeLa cells was formed on the bottom of the Corning flasks, which was evidence of the high viability of HeLa cells. On the second day, the bottom of the Corning flasks was covered with a monolayer of HeLa cells and all subsequent experiments were conducted using this material.

Figure 1 shows HeLa cells grown during 2 days as a monolayer on the bottom of Corning flasks. At a magnification of 400 times, intact HeLa cells could be observed, characterized by thick cell walls and large nuclei containing 1–3 nucleoli.

As shown in Figure 1(2), the dye NBT stained HeLa cells with functional mitochondria, resulting in a purple-blue coloration. Conversely, the indicator of dead cells with damaged membranes, trypan blue (TB) (Figure 1(3)), did not stain the original HeLa cells.

Figure 1(2) illustrates the outcome of the action of HPV16 E2 on a two-day suspension of HeLa cells obtained from the monolayer on the bottom of corning flasks. The cells were suspended in DMEM medium supplemented with antibiotics.

Immediately after adding 30 μl of HeLa cell suspension to the supernatant of the homogenate of transgenic tomato (containing HPV16 E2) and 20 μl of 0.4% trypan blue (TB), staining of the HeLa cells was observed, accompanied by flotation due to disruption of adhesion to the glass slide (Figure 2(1)). After 30 minutes, these free-floating HeLa cells aggregated into flakes, eventually forming tightly dyed lumps (Figure 2(2)). Subsequent changes in the condition of the stained HeLa cells with TB resulted in the compaction of these floating cells (Figure 2(3)).

It was discovered that the blood serum of mice vaccinated with the vaccine material derived from transgenic tomato fruit containing the HPV16 E2 gene had a similar effect as the transgenic protein HPV16 E2 itself. This serum facilitated the penetration of trypan blue (TB) into HeLa cells with damaged membranes, resulting in the aggregation of HeLa cells into tightly dyed lumps (Figure 3(1) and (2)).

However, during the exposure period, the structured conglomerates of stained cells began to undergo changes, gradually forming various dendritic forms in some areas and losing their color (Figure 3(3–6)), despite the entire slide being loaded with TB. Notably, no such transformation was observed after the action of blood serum from non-vaccinated native mice on HeLa cells (data not presented).

After the addition of LAAO (30 μl of a 10 mg/ml solution), tightly stained flakes of HeLa cells formed in the presence of TB within the first 30 minutes (Figure 4(1)). Subsequently, after 2 hours (Figures 4(2)) and 3 hours (Figure 4(3)), there was a reduction in color intensity and the compactization of HeLa cells into more organized structures. By 4 hours, more than half of the HeLa cells had become colorless and densely compacted in a monolayer, with approximately 30% of the remaining cells remaining unpainted with TB (Figure 4(4)).

The metamorphosis of HeLa cells following the addition of 10 μl of a 10 mg/ml solution of LAAO from the North American rattlesnake, Crotalus adamanteus, along with 30 μl of TB, is presented in Figure 5. Within the first 5–30 minutes, stained blue conglomerates of HeLa cells formed (Figure 5, upper row). Subsequently, these clusters began to exhibit dichotomic branching and appeared as colorless branches during the subsequent hours of exposure to the snake venom solution with TB (Figure 5, middle row). After 5 hours, all HeLa cells were observed in the form of a continuous symplast, resembling bilayer membranes in three dimensions with branching without subdivision into individual cells (Figure 5, lower row).

From Figure 5 (final lower row), it is apparent that single HeLa cells were not observable under the light microscope even at a magnification of 1000 times. This suggests the possibility of membrane fusion of all cells into a uniform, continuous symplast without cytokinesis under the action of LAAO. It can be speculated that these structures serve functions of protection and sustainable self-preservation for HeLa cells. The dimensions of this symplast can be estimated based on the dimensions of the Goryaev chamber, where the distance between two vertical lines (Figure 5, middle row, photo in the center) amounted to 1/1600 mm².

The addition of DAAO (from porcine kidney, Sigma, USA) to HeLa cells resulted in the formation of unusual structures (Figure 6). Upon the addition of TB together with DAAO, there was a loss of adhesion, and individual cells floated in the suspension of HeLa cells with DMEM. Subsequently, the HeLa cells began to conglomerate, and after 5 hours of exposure, the cellular structures transformed into a tightly compacted single grainy layer (Figure 6).

It was observed that during the action of the “early” protein HPV16 E7 in the presence of TB, HeLa cells lost adhesion and became suspended within the first few minutes of addition but remained unstained (Figure 7).

The phenomenon of flotation and disruption of monolayer growth is typically considered an indication of aging in HeLa cells. Some of these cells showed slight staining with TB after 3 hours of exposure. Slow and weak staining was observed with HPV16 E6 in the presence of TB (data not presented).

Since oncoproteins HPV16 E6 and E7 do not possess the supersuppressor-oncolytic activity observed with HPV16 E2, these proteins may not activate the instability of HeLa cell membranes. Hence, staining occurred slowly (Figure 7).

CAS was used to excise oncogenes E6 and E7 from HeLa cells. Endonuclease CAS targeted these oncogenes using a guide RNA with a template derived from RdRP. This decision was made based on the assumption that multiple genomes of related papillomavirus HPV18 were present in HeLa cells (Figure 8).

3.2 Unusual “alien” structures found in the abdomens of mice infected with Hela cells sensitive to HPV16 E2

In addition to the unusual behaviors observed in HeLa cells under stress conditions, unique structures were discovered in the abdomens of mice (Figure 9). Male mice were initially infected with HeLa cells by injecting 100 μl of the cells into the lateral thigh muscle. Subsequently, after 1 month, they were vaccinated orally three times with an interval of 1 month using vaccine material transgenic with HPV16 E2.

“Alien residents” manifested as coral-like structures that were fragile and lacked any discernible internal structural elements. Occasionally, one or two of these “aliens” were observed in the mouse gut, unattached to the abdomen. The traces of blood seen in Figure 9 were from the wound incurred during abdominal dissection, indicating that these structures likely developed autonomously.

Another peculiar finding frequently observed in the abdomens of infected mice were spheres of white or pearl color, measuring 1.5–1.8 cm in diameter. These spheres rapidly shrunk in size upon exposure to air or DMEM medium, and fully dissolved upon addition of supernatant from buffer homogenates of tomato fruit containing HPV16 E2 (Figure 10).

Figure 10 depicts the pearl-colored spheres observed upon opening the abdomens of mice infected with HeLa cells. No connecting elements were identified on the inner surface of the abdominal cavity, nor were there any blood vessels or traces of blood present. It is presumed that the development of these spheres occurred autonomously.

Following investigations conducted with HeLa cells in vitro, it was deemed necessary to examine the effects of HPV16 E2, LAAO, and DAAO on mice lung tumors ex vivo using a model of T lymphocytes isolated from spleens infected with HeLa cells.

3.3 The “education” of “naive” mice splenocytes after incubation in suspension of HeLa cells on tumor growths in mice to force tumors into regression

Non-effector (inactive and unloaded with antigen) “naive” macrophages, T lymphocytes, and splenocytes are powerful factories of protein biosynthesis, particularly in terms of synthesizing receptors on the surfaces of lymphocytes. It is estimated that approximately 100,000 different types of receptors may be present on the surfaces of lymphocytes. Therefore, it can be assumed that mice T lymphocytes and splenocytes can be trained to serve as effectors bearing receptors capable of recognizing tumor antigens. The number of T lymphocytes in an adult organism may reach as much as 10¹⁶ cells.

The training, or “education,” of T lymphocytes typically occurs in their native habitat, such as the bone marrow or thymus. Antigens of host proteins are presented to naive T cells to eliminate those lymphocytes that might potentially react with host antigens. Those lymphocytes that respond after recognition are eliminated.

The aim of the experiment was to train splenocytes, predominantly naive ones, isolated from the spleen of native mice by exposing them to a suspension of HeLa cells for 5 days. After 5 days, the fractions of splenocytes trained with HeLa cells were placed in cultivated 24-well plates with lung tumor growths caused by HeLa cells during their two-day incubation with HeLa cells.

Five mice (3 females and 2 males, aged 7 months, with masses of 35–40 g) were used in the experiment. Lungs and spleens were immediately placed into a two-day suspension of HeLa cells (taken from a low-temperature freezer) in cultivating Corning flasks (25 ml) with DMEM medium containing 500 units of penicillin and 50 mg/l streptomycin. Membrane nitrocellulose disks with a diameter of 14 mm were placed into wells of 5 plates with 24 wells each, with each plate corresponding to a different experimental variant: (1) DMEM only, (2) HPV16 E2 as the activator, (3) LAAO as the activator, (4) DAAO as the activator, and (5) HPV16 E2 as the activator (replication of variant 2). Each well contained 300 μl DMEM, 30 μl of splenocyte fraction, lungs or their tumor fragments, and 300 μl of each activator. The plates were covered with lids, packed into plastic films, and left for 5 days at room temperature.

After 5 days, the disks in the wells were washed three times with TNT buffer, and relevant antibodies (IgG) targeting interferon, CD4/CD8 T lymphocytes, T cell receptors, and apoptotic enzymes (granzyme B, perforin, and granulysin) were added. Incubation lasted for 1 day, followed by washing the plates three times with TNT buffer. Secondary antibodies to mice IgG conjugated with alkaline phosphatase were then added. After another day of incubation, the plates were washed with TNT buffer, dried in the air, and the substrate BCIP (or NCIP)/NBT was used to develop spots of colonies of immunopositive T cells.

The incubation of isolated lungs resulted in extensive tumor growths, as depicted in Figure 11 (left and center). These growths appeared as cellular tumor neoplasms of light color in the central part and partly on the periphery of the inoculated lungs.

However, keeping the spleens in the suspension of HeLa cells did not lead to any visible changes in the surface, color, volume, or structure of the spleens, as shown in Figure 11 (right).

It appears that the “trained” splenocytes induced the regression of tumor lungs independently of the presence of activators such as oncolytics HPV16 E2, LAAO, and DAAO, as shown in Figure 12.

3.4 Elispot analyses of immunogenicity of “educated” splenocytes

Despite the well-known oncolytic and antiproliferative activity of LAAO and DAAO [27, 28, 29], it was decided to investigate their effects on the immunogenicity of components of the T-cell unit in mice. This included studying their action on peripheral mononuclear blood cells (PMBCs), splenocytes, and lungs inoculated with HeLa cells, as illustrated in Figure 13.

The results shown in Figure 13 demonstrate that LAAO and DAAO significantly increased the synthesis of interferon and apoptotic enzymes (granzyme B, perforin, and granulysin) in both PMBCs and splenocytes. The counts of inducible immunopositive cells were notably higher than the control groups, showing an increase of up to two times or more.

Furthermore, the induction of components of the T-cell response in mice lungs, which were incubated with HeLa cells for 5 days, yielded intriguing results. Mice lungs incubated with HeLa cells for 2 days developed tumor growths, specifically small cell sarcomas. T lymphocytes isolated from these tumor-bearing lungs were subjected to Elispot analysis using antibodies against interferon and apoptotic enzymes (granzyme B, perforin, and granulysin), as depicted in Figure 14.

The results of Elispot analyses (Figure 14) clearly demonstrate the activation of the antitumor defense mediated by T lymphocytes in the lungs of mice bearing tumors treated with HeLa cells. This activation is evidenced by a significant increase in the synthesis of interferon and apoptotic enzymes, including granzyme B, perforin, and granulysin. Numerous colonies of immunospots generated by T lymphocytes, well stained and dense, were observed.

It was hypothesized that T lymphocytes from the lungs of mice infected with HeLa cells were capable of being “trained” by internal antitumor mechanisms induced during the infection. Based on the results of Figures 12 and 14, it could be assumed that splenocytes and T lymphocytes were also “trained” by similar internal antitumor mechanisms, likely components of the microenvironment.

To test this assumption, experiments were conducted in which spleens and lungs of mice were infected with HeLa cells for 5 days, resulting in tumors on the lung surfaces while leaving the spleens visibly unchanged. Subsequently, splenocytes were isolated from the treated spleens and placed into wells containing tumor-bearing mice lungs (or fragments thereof), with nitrocellulose membrane disks. Additionally, activators such as HPV16 E2, LAAO, and DAAO were added. Controls included wells with only nitrocellulose membrane disks, supplemented with DMEM with antibiotics and 20 μl of isolated splenocyte fraction.

Intact splenocytes from untreated spleens, isolated from mice infected with HeLa cells injected into the lateral thigh muscle for 2 months, showed a weak immune response. Only a few T lymphocytes reacted with the corresponding antibodies to T cell receptor (TCR) and apoptotic enzymes (granzyme B, granulysin, and perforin). Activated colonies of T lymphocytes, numbering between 200 and 500 immunospots on nitrocellulose membrane disks, were found.

Therefore, it was decided to investigate the effects of oncolytics (HPV16 E2, LAAO, and DAAO) on isolated splenocytes from spleens inoculated with a suspension of HeLa cells for 5 days. Five mice, comprising 3 females and 2 males, aged 7 months and weighing between 30 and 40 g, were selected for analysis.

Based on the characteristics of immunospots on nitrocellulose membranes and the results of counting their numbers (Figure 15), it can be inferred that a significant and robust immune response occurred in spleens inoculated with HeLa cells, indicating a strong impact in response to the presence of HeLa cells. The number of immunospots increased by a factor of 10 compared to the number detected on membrane nitrocellulose disks visible in Figure 16. Furthermore, an additional increase in the number of immunospots was observed in the presence of activators such as HPV16 E2, LAAO, and DAAO. Particularly noteworthy was the pronounced impact of LAAO and DAAO, with the number of immunospots increasing by 20–30 times compared to those represented on the membrane nitrocellulose disks in Figure 16.

These findings are consistent with the observed regression of lung tumors in mice in the presence of HPV16 E2, LAAO, and DAAO (Figure 12), demonstrating a correlation between the immune response observed in the spleens and the therapeutic effects on tumor regression induced by these agents.