RU2759487C1 - Method for enhancing the growth of b16/f10 melanoma versus the growth of b16/f10 melanoma with self-grafting and slowing the growth of llc (lewis carcinoma) versus the growth of llc with self-grafting in case of multiple primary malignant tumors against the background of primary immunodeficiency - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and relates to a method for enhancing the growth of B16 / F10 melanoma in comparison with the growth of B16/F10 melanoma with self-inoculation and slowing down the growth of LLC (Lewis carcinoma) compared with the growth of LLC with self-inoculation in primary multiple malignant tumors against the background of primary immunodeficiency , which consists in the fact that female mice of the BALB/cNude line are successively inoculated under the skin of the back from both sides with two tumor strains: a suspension of tumor cells B16/F10 and LLC.

EFFECT: creation of experimental model of B16/F10 and LLC growth in mice for studying the pathogenesis of multiple primary malignant tumors.

1 cl, 1 ex, 1 tbl

Description

The invention relates to oncology, namely to experimental oncology, and can be used to study the primary multiple process in an experiment.

Primary Immunodeficiency Disorders — Primary Immunodeficiency Disorders (PID) are a group of more than 400 inherited diseases that occur when at least one component of the immune system is absent or dysfunctional (see Bousfiha A. et al., 2019; Tangye S. G. et al., 2020) ... Patients with PID have a higher risk of developing severe and often fatal infections, allergies, autoimmune complications, and malignant neoplasms (see Haas O.A., 2018; deWitJ. Etal., 2019). Not so long ago, PID was considered a rare condition. However, with the advent of next-generation high-throughput sequencing technologies, the accuracy of PID diagnostics has improved markedly (see GalloV. Et al., 2016), as a result, the PID frequency has increased from about 1/10000 to 1 / 1000– 1/5000 in recent years (see TangyeS . G. etal., 2020). The discovery of new genetic defects continues to increase the frequency of PID. For these reasons, a better understanding of the complications associated with PID is critical to improving patient management and clinical outcomes.

Among the complications associated with PID, special attention is paid to cancer. Several large cohort studies conducted in different parts of the world have shown an increased relative risk of cancer in patients with PID (see Jonkman-Berk B.M. et al., 2015; Mayor P.C. et al., 2018). PID cancers often include hematologic malignancies, which may arise from an intrinsic inability to regulate genetic instability and cell transformation during hematopoietic cell differentiation and / or from altered immune surveillance of the tumor by cytotoxic lymphocytes. During B cell receptor (BCR) and T cell receptor (TCR) diversification, B and T cells undergo complex genetic changes, including double-stranded DNA breaks, DNA repair, cell proliferation, and cell death (AltF.W. et al., 2013).

Immunological surveillance of the tumor is carried out by several types of immune cells with cytotoxic lymphocytes, CD8 ₊ T cells and natural killer (NK) cells, which play an important role in the elimination of cancer cells (Martinez-Lostao L. et al., 2015). The detection and destruction of cancer cells depends on the ability of cytotoxic lymphocytes to form a stable and functional lytic immunological synapse (IS).

IS can be defined as the interface between a cytotoxic immune cell and a target cell. The IS center consists of a closed nanoscale space called the synaptic cleft, where most of the intercellular communication occurs. Both the cytotoxic cell and the target cell interact with a variety of complementary receptors and ligands through the synaptic cleft. For CD8 + T cells, the interaction between the T cell receptor (TCR) and the class I major histocompatibility complex (MHC) is critical to killing the target cell.

For NK cells, recognition of activating and inhibiting ligands by receptors specific for NK cells results in a killing response. These interactions trigger intracellular signals that allow the cytotoxic lymphocytes to respond appropriately to the cell they encounter. When a related target cell is a healthy cell, an inhibitory synapse is formed. This inhibitory synapse prevents the involvement of the lytic apparatus of cytotoxic lymphocytes and thus protects healthy cells from cytotoxic deleterious effects (Wurzer H. et al., 2019). When cytotoxic cells contact antigen presenting cells (APCs) such as dendritic cells (DCs), the lytic cascade is not triggered and the assembled synapse is called a regulatory synapse (WurzerH. Et al., 2019). Regulatory synapses are required to stimulate the priming of CD8 + T cells by DCs, but also to license DCs by CD4 + T cells. Finally, if the target cell is recognized as malignant, lytic IS is formed (Wurzer H. et al., 2019).

A characteristic feature of lytic IS is the secretion of cytotoxic molecules within the cleft. Once released, cytotoxic molecules enter cancer cells and cause their death, mainly through apoptosis. The targeted release of cytotoxic granules towards the target cell ensures that the surrounding healthy cells are not affected by the protective mechanism of immune surveillance (HsuH.T. et al., 2016). In PID patients with abnormally functional cytotoxic lymphocytes, immune surveillance is impaired (Bousfiha A. et al., 2019; Tangye S. G. etal., 2020). As a result, malignant neoplasms often develop and progress in patients with PID. Dysfunctions of cytotoxic cells can occur at several stages during the destruction of cancer cells and can affect several subcellular networks. For example, IS often changes in patients with PID. In most cases, the loss of molecules in the actin and cytoskeleton of microtubules causes defective IS formation. Actin and microtubular cytoskeleton are required to support the architecture and dynamics of IS, especially the three concentric supramolecular activation clusters (SMACs) (central, peripheral and distal) seen in IS T cells (Hammer J.A. et al., 2019). In addition to mutations in proteins of the cytoskeleton, mutations in molecules of the exocytosis apparatus are usually found in patients with PID.

Over the past decade, the advances and availability of genetic diagnostic tools such as next generation sequencing have shed light on the prevalence of PID with approximately 400 identified abnormalities (Bousfiha A. et al., 2019). Also, large multicenter studies were conducted in different countries to describe the complexity of clinical manifestations (Abolhassani H. et al., 2018). Systematic reviews of epidemiological data describing the incidence and nature of malignant neoplasms associated with PID have produced surprising findings. First, in patients with PID, malignant neoplasms occur more often and earlier (RiazI.B. Et al., 2019). Second, the nature of malignant neoplasms associated with PID is different from malignant neoplasms caused by secondary immunosuppression. Most malignant neoplasms manifest as melanoma and renal cell carcinoma (Hauck F. et al., 2018).

Due to a weakened immune system, PID patients cannot effectively eradicate the pathogens that contribute to the development of cancer. The immunocompromised environment of PID patients cannot successfully stop the spread of cancer-promoting pathogens and is unable to effectively eliminate tumor cells as soon as they appear. In patients with PID, the process of "searching and destroying" tumors, organized by cytotoxic CD8 + T cells and NK cells, is often defective. The absence of both CD8 + T cells and NK cells, as described in some severe combined immunodeficiencies, can easily make immune surveillance of the tumor nearly impossible. However, some changes usually found in patients with PID are less noticeable in the sense that they interfere with the function of cytotoxic lymphocytes, rather than simply hinder their development. This is especially true for genetic mutations with negative consequences for the formation, stability or function of IS (Mastio J. et al., 2020).

Cancer has now become a serious threat to human health. In various studies from different countries, it was reported that the incidence of primary multiple malignant tumors varied from 0.52% to 11.7% (Demandante C.G. et al., 2007; Lv, M.V. et al., 2017). The incidence, characteristics and survival rates associated with multiple primary malignant tumors (PMTCs) varied across geographic regions. It is reported that in 2015 there were about 4,292,000 newly diagnosed cancer cases in China, which corresponds to almost 12,000 new cancer diagnoses on average every day (Chen W. et al., 2016). In the United States, there were a total of 1,658,370 new cancer cases in 2015 (Siegel R.L. et al., 2015). A study of patients who underwent a full-body PET / CT scan showed that at least 1.2% of patients with cancer had another primary malignant tumor (Ishimori T. et al., 2005). The likelihood of developing synchronous malignant neoplasms varies from 34.9 to 41%, and for metachronous neoplasms from 59 to 66% in different studies (Sapalidis, K. et al., 2016). A meta-analysis based on literature sources shows that the incidence of a second tumor is 3 to 5%, a third tumor is about 0.5% and a fourth tumor is 0.3% (Sisti A. et al., 2016).

To study the mechanisms of carcinogenesis, it is important to choose an appropriate model. To date, the most commonly used mouse and rat models. The mouse has a number of advantages over other mammals: financially beneficial housing, short reproductive cycle, and the ability to use genetic engineering methods. The line of mice BALB / c (Bagg albino C) and C57Bl / 6J - an inbred line of black color, is used in almost all medical and biological research. Both of the above lines are standard for oncology. Also, for the work of immunologists and oncologists, mice with suppressed immunity are required. These mice lack the thymus, which forms their primary immunodeficiency, and hair. They are known as nude mice.

A known method of reproducing liver metastases in an experiment by inoculating a malignant tumor into the spleen of white outbred rats, previously removed under the skin. After the surgical wound has healed, a suspension of tumor cells of sarcoma 45 in saline in a volume of 0.1 ml at a dilution of 1 × 10 ^{6 is} implanted into the spleen tissue. After 2.5-3 weeks, a morphological examination of the liver is carried out. The proposed method eliminates the influence of stress factors (surgery, general anesthesia) on the process of metastasis of a malignant tumor to the liver, bringing it closer to natural; allows you to track the growth dynamics of the primary tumor node, assumes the spread of metastases to the liver only in the most significant - hematogenous way, and can be used to develop methods of experimental therapy for this pathology (see RF Patent No. 2538243).

There is a method of modeling lymphogenous and hematogenous metastasis of murine melanoma B16 / F10 in white nonlinear rats, which consists in the fact that 2 weeks after preliminary removal of the spleen under the skin of the anterior abdominal wall, a suspension of tumor cells of murine melanoma B16 / F10 in saline in volume 0.1 ml and at a dilution of 1:10, and six months after the inoculation, the lymphogenous and hematogenous pathways of melanoma spread with morphologically confirmed metastatic lesions of internal organs: liver, thymus, lymph nodes, pancreas are recorded (see RF Patent No. 2615908).

A known method for obtaining a malignant tumor growing in lung tissue. A suspension of tumor cells before administration is diluted with sterile saline 1: 5 by volume and drawn into a sterile syringe. Male white outbred rats, after fixing them on the back, are injected with a tumor suspension into the subclavian vein at a dose of 0.5 ml. On days 14-21 from the moment of inoculation of sarcoma 45, a visualized tumor develops in males, which can be removed and transplanted. According to the authors, this method should provide 100% reproducibility, leading to the inoculation of tumor grafts in all cases, which, in turn, will make it possible to conduct mass experiments in a relatively short time and in large quantities with ease of execution (see RF Patent No. 2388064) ...

A known method for obtaining a malignant tumor,
growing in the lung tissue by introducing benzpyrene into the subclavian vein in male white outbred rats in a dose of 0.5 ml. The tumor develops after 4-4.5 months. This model is convenient for studying the pathogenesis of malignant growth. The model makes it possible to set up mass experiments with simplicity of execution (see Patent No. 2375758).

Lung carcinoma LLC arose spontaneously in C57Bl / 6 mice in 1951, the proliferative pool at the time of tumor grafting - 73.8% (see AM Kozlov, ZP Sof'ina, 1978). Melanoma B16 arose spontaneously at the base of the ear in the skin of a C57BL / 6 mouse in 1954. With transplantation under the skin, the development of the tumor is 100% (see Fadeeva, E.V., 2010).

The technical result of the present invention is the creation of an experimental model of the growth of B16 / F10 and Lewis lung carcinoma (LLC) in BALB / c Nude mice to study the pathogenesis of multiple primary malignant tumors (PMZO).

The technical result is achieved by the fact that female mice of the BALB / cNude line are successively inoculated under the skin of the back on both sides of two tumor strains: 0.5 ml of a suspension of tumor cells of mouse melanoma B16 / F10 in saline at a dilution of 1:10, on the right and LLC 2 million cells in 0.5 ml of saline, on the left; at the same time, after removing the needle, the injection site is tightly pressed with a cotton swab dipped in 70% alcohol with the addition of iodine for 1 minute in order to exclude leakage of tumor material.

The invention "A method for modifying malignant growth in primary multiple malignant tumors against a background of primary immunodeficiency" is new, since it is unknown in the field of experimental oncology about changes in the growth of B16 / F10 and LLC melanoma in BALB / c Nude mice to study the pathogenesis of polyneoplasias under conditions primary immunodeficiency.

The novelty of the invention lies in the use of female BALB / c Nude mice to create a PMZO model under conditions of primary immunodeficiency, in which the development of a combined malignant process: melanoma B16 / F10 and LLC in the body follows a non-standard “scenario”. Reproduction of the PMZO model by subcutaneous inoculation of B16 / F10 melanoma and LLC into BALB / c Nude mice changes the growth rate of both tumors compared to self-inoculation: it enhances the growth of B16 / F10 and slows down LLC.

The invention "A method of enhancing the growth of melanoma B16 / F10 in comparison with the growth of melanoma B16 / F10 with self-inoculation and slowing the growth of LLC (Lewis carcinoma) compared with the growth of LLC with self-inoculation in primary multiple malignant tumors against the background of primary immunodeficiency" is industrially applicable , since it can be used in oncological research institutions to reproduce an experimental growth model of two morphologically dissimilar malignant tumors, to study their growth, pathogenesis, and search for ways to influence them.

"The method of enhancing the growth of melanoma B16 / F10 compared with the growth of melanoma B16 / F10 with self-inoculation and slowing the growth of LLC (Lewis carcinoma) compared with the growth of LLC with self-inoculation in primary multiple malignant tumors against the background of primary immunodeficiency" is carried out as follows.

Female mice of the BALB / c Nude line under the skin of the back just below the right shoulder blade are injected with 0.5 ml of a suspension of tumor cells of mouse melanoma B16 / F10 in saline at a dilution of 1:20, on the other hand, just below the left shoulder blade, 0.5 ml is injected subcutaneously tumor suspension LLC containing 0.5 million tumor cells. To do this, observing all aseptic conditions, the assistant fixes the mouse with its back up, having previously treated the skin with a 5% alcohol solution of iodine. The experimenter with a sterile gloved hand grasps the skin fold, in the center of which he pierces the skin and injects a tumor suspension B16 / F10 on one side, and LLC on the other. After removing the needle, the injection site is tightly pressed with a cotton swab dipped in 70% alcohol with a small addition of iodine for 1 minute to prevent leakage of tumor material. Controls are female BALB / c Nude mice with self-inoculation of either B16 / F10 melanoma or LLC in the same dose and volume.

Features of tumor growth during independent and combined tumor grafting are presented in the table.

table

Tumor volume and lifespan of female Nude mice

with PMZO

melanoma B16 / F10 Lewis carcinoma PMZO melanoma B16 / F10 Lewis carcinoma tumor volume
for 15 days, cm3 0.82 ± 0.22 0.94 ± 0.13 2.31 ± 0.54 ^* 0.42 ± 0.11 ^{*, +, 1} tumor volume
for 22 days, cm3 3.90 ± 0.86 4.74 ± 0.80 6.89 ± 0.94 ^* 2.03 ± 0.21 ^{*, +, 1} life expectancy, days 26.00 ± 0.58 28.67 ± 0.33 22.67 ± 0.88 ^{*, +}

Note: ^* - significance of differences in comparison with isolated growth of melanoma, ⁺ - significance of differences in comparison with isolated growth of Lewis carcinoma, ¹ - significance of differences in Lewis carcinoma from melanoma in the PMZO model.

As can be seen from the results of the table, the tumors inoculated in an independent variant: melanoma B16 / F10, or LLC, on the 15th day after inoculation had practically the same volume of the tumor node. In mice with combined inoculation (PMZO model) on day 15, the tumors had different volumes, which differed from the volume of tumors with self-inoculation: the volume of B16 / F10 melanoma with combined inoculation was 2.8 times greater than with independent, and the volume of LLC, on the contrary , 2.2 times less. At the same time, the volume of B16 / F10 melanoma during this study period was 5.5 times greater than the LLC volume (see Table). On the 22nd day after inoculation, the volume of B16 / F10 melanoma with combined inoculation was 1.8 times (p <0.05) more than with independent inoculation, and the volume of LLC, on the contrary, was 2.3 times less. At the same time, the volume of B16 / F10 melanoma during this study period was 3.4 times greater than the LLC volume, and with self-inoculation did not have statistically significant differences (see Table).

Thus, combined subcutaneous inoculation of B16 / F10 and LLC melanoma into BALB / c Nude mice increased the malignant potential of melanoma and decreased the malignant potential of LLC, which was accompanied by a decrease in the lifespan of the animals as compared with the individual inoculation of each tumor.

Technical and economic efficiency "A method of enhancing the growth of B16 / F10 melanoma in comparison with the growth of B16 / F10 melanoma with self-grafting and slowing the growth of LLC (Lewis carcinoma) compared with the growth of LLC with self-grafting in primary multiple malignant tumors against the background of primary immunodeficiency." lies in the fact that the reproduction of the PMZO model under conditions of primary immunodeficiency by sequential subcutaneous inoculation of melanoma B16 / F10 and LLC into BALB / c Nude mice changes their malignant potential: it activates the growth of B16 / F10 and slows down the growth of LLC. This makes it possible to study the pathogenesis of a synchronous malignant process in tumors of various histological structures and the effect of comorbid pathology on them, such as primary immunodeficiency, as well as to search for targets for targeted therapy of PMZO. The method is economical and available for execution.

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Claims (1)

A method of enhancing the growth of B16 / F10 melanoma compared with the growth of B16 / F10 melanoma with self-grafting and slowing the growth of LLC (Lewis carcinoma) compared with the growth of LLC with self-grafting in primary multiple malignant tumors against the background of primary immunodeficiency, which consists in the fact that female mice of the BALB / cNude line are successively inoculated under the skin of the back on both sides with two tumor strains: 0.5 ml of a suspension of tumor cells of murine melanoma B16 / F10 in saline at a dilution of 1:10 on the right and LLC 2 million cells in 0.5 ml of physiological solution on the left; in this case, after removing the needle, the injection site is tightly pressed with a cotton swab dipped in 70% alcohol with the addition of iodine for 1 minute in order to exclude the leakage of tumor material.