CC BY
0South Russian Journal of Cancer. 2024. Vol. 5, No. 1. P. 25-33 https://doi.org/10.37748/2686-9039-2024-5-1-3 https://elibrary.ru/kcljwh
ORIGINAL ARTICLE
Evaluation of engraftment and growth dynamics of orthotopic
and heterotopic in vivo models of human breast cancer
I. S. Lyashenko, M. V. Romanova, A. S. Goncharova12, D. V. Khodakova, A. V. Galina, S. V. Gurova,
S. Yu. Filippova, Yu. S. Shatova
National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation
12 fateyeva_a_s@list.ru
Purpose of the study. This work was to assess the engraftment and growth dynamics of breast cancer xenografts during orthotopic and subcutaneous injection using various types of biological material, as well as to develop an adequate model of breast cancer for further research.
Materials and methods. We used a disaggregated fragment of a tumor obtained from the patient, a certified breast cancer cell line VT20 - human breast carcinoma; a primary human breast carcinoma cell line. Female immunodeficient mice of the Balb/c Nude line in the amount of 36 animals were used as recipient animals. The subcutaneous and orthotopic models of breast cancer were developed in this project. Tumor growth was observed for 28 days from the moment of injection and tumor nodes were measured 2 times a week until the end of the experiment. Results were assessed using medians and percentiles. The nonparametric Mann-Whitney test was used to assess the significance of differences.
Results. The dynamics of the growth of tumor cells when injected into various sites was determined in the process of this work. The most successful in terms of a subcutaneous injection was the injection of tumor cells of the certified VT20 line. By the end of the experiment, the median tumor node of this group was 100.32 mm�. The analysis revealed tumor dynamics with orthotopic injection of tumor material, and the median volume of the tumor node in the group with the passport culture cell VT20 and the primary culture cell reached the same value - 149.22 and 148.25. mm�. It was found that both the cell line and the cell suspension were injected into tumor nodes that reached a significantly larger volume when injected orthotopically. Conclusion. We have obtained a tumor model of breast cancer using various methods of material implantation and with the possibility of further use in testing new pharmacological substances.
For citation: Lyashenko I. S., Romanova M. V., Goncharova A. S, Khodakova D. V., Galina A. V., Gurova S. V., Filippova S. Yu., Shatova Yu. S. Evaluation of engraftment and growth dynamics of orthotopic and heterotopic in vivo models of human breast cancer. South Russian Journal of Cancer. 2024; 5(1): 25-33. (In Russ.). https://doi.org/10.37748/2686-9039-2024-5-1-3, https://elibrary.ru/kcljwh
For correspondence: Anna S. Goncharova - Cand. Sci. (Biol.), head of testing Laboratory Center, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation
Address: 63 14 line str., Rostov-on-Don 344037, Russian Federation E-mail: fateyeva_a_s@list.ru
ORCID: https://orcid.org/0000-0003-0676-0871 SPIN: 7512-2039, AuthorID: 553424
Scopus Author ID: 57215862139
Compliance with ethical standards: All manipulations during the experiment were performed in compliance with the ethical principles established by the European Convention for the Protection of Vertebrate Animals Used for Experiments or Other Scientific Purposes (ETSN 123, Strasbourg, March 18, 1986). Study Protocol No. 19/123 dated 08/3/2021 was approved by the local Ethical committee National Medical Research Centre for Oncology. The patient provided written consent for the transfer of biological material
Funding: this work was not funded
Conflict of interest: the authors declare that there are no obvious and potential conflicts of interest associated with the publication of this article
The article was submitted 08.09.2023; approved after reviewing 10.02.2024; accepted for publication 27.02.2024
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??????????. ? ???? ?????? ?????? ???? ?????????? ???????? ????? ?????????? ????? ??? ???????? ? ????????? ?????. ???????? ????????, ??? ????????? ????????, ???????? ???????? ?????????? ??????, ?????????????????? ????? ?? 20. ?? ?????? ????????? ???????????? ??????? ?????????? ????? ?????? ?????? ????????? 100,32 ??�. ??? ??????? ???????? ????? ??????? ??? ?????????????? ???????? ??????????? ????????? ???? ????????, ??? ? ??????? ?????? ?????????? ????? ? ?????? ? ?????????????????? ????????? ?????? ??20 ? ????????? ????????? ?????? ????????? ??????? ???????? - 149,22 ? 148,25 ??�. ???? ????????, ??? ??? ??? ???????? ????????? ?????, ??? ? ????????? ????????? ?????????? ???? ????????? ??????? ???????? ?????? ??? ?????????????? ????????. ??????????. ???? ???? ???????? ?????????? ?????? ??? ??? ????????? ???????? ??????????? ????????? ? ? ???- ????????? ??????????? ????????????? ??? ???????????? ????? ????????????????? ??????????.
??? ???????????: ??????? ?. ?., ???????? ?. ?., ????????? ?. ?., ???????? ?. ?., ?????? ?. ?., ?????? ?. ?., ????????? ?. ?., ?????? ?. ?. ?????? ??????????? ? ???????? ????? ?????????????? ? ???????????????? in vivo ?????? ???? ???????? ?????? ????????. ????-?????????? ?????????????? ??????. 2024; 5(1): 25-33. https://doi.org/10.37748/2686-9039-2024-5-1-3, https://elibrary.ru/kcljwh
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?????: 344037, ?????????? ?????????, ?. ??????-??-????, ??. 14-? ?????, ?. 63 E-mail: fateyeva_a_s@list.ru
ORCID: https://orcid.org/0000-0003-0676-0871 SPIN: 7512-2039, AuthorID: 553424
Scopus Author ID: 57215862139
?????????? ????????? ??????????: ??? ??????????? ? ???? ???????????? ???? ????????? ? ??????????? ????????? ?????????, ????????????? ??????????? ?????????? ? ?????? ??????????? ????????, ???????????? ??? ????????????? ??? ? ???? ??????? ????? (ETSN 123, ?????????, 18 ????? 1986 ?). ???????? ???????????? ? 19/123 ?? 3.08.2021 ?. ??? ??????? ????????? ????????? ????????? ???? "???? ?????????" ????????? ??????. ?????????? ???? ????????????? ?????????? ???????? ?? ???????? ?????????????? ?????????
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INTRODUCTION
Breast cancer (BC) is one of the most common cancers in the world. So in 2022, about 68 thou- sand new cases of this disease were registered in Russia [1]. According to global statistics, the inci- dence of BC is 11.6 % of all cancer cases, and about 626,000 deaths from BC, which accounted for 6.6 % of reported deaths [2]. In addition, this disease is highly heterogeneous and is divided into 4 differ- ent molecular subtypes, differing in the genomics of the tumor and the type of cells from which the tumor is initiated. So these subtypes include lumi- nal A, luminal B, Her2-positive and triple negative BC [3]. To study different subtypes, scientists use several types of model systems: in silico, in vitro and in vivo [4]. Each of these methods has a num- ber of advantages and disadvantages. Using the in silico model system, scientists are searching for potential target antigens and analyzing transcrip- tomic data [5], analysis of the relationship between genotype and phenotype to search for a gene as a promising target for therapy, as well as the search for directly or indirectly related genes selected as an alternative to the found target [6] and computer prediction of the complementarity of the binding region between the drug and the therapeutic target against BC [7]. When using the in vitro system, re- searchers analyze the mechanisms of resistance of tumor cells to drugs, evaluate the effect of new pharmacological substances on the viability of tu- mor cells [8], the mechanisms of organotoxicity of drugs are being investigated [9] and so on. Currently, the development of BC tumor models in vivo is also relevant in order to study biomarkers of tumor sen- sitivity to drugs, study new treatment regimens, and study the development of tumors in the body [10].
The purpose of the study was to evaluate the en-
graftment and growth dynamics of BC xenografts during orthotopic and subcutaneous implantation us- ing various types of biological material, as well as to develop an adequate BC model for further research.
MATERIALS AND METHODS
Tumor material
For this work, we used a disaggregated fragment of a tumor obtained from a patient E. 75 years old, with digested breast cancer cT4N2Mo St IIIB, a cer-
tified BC VT20 cell line - human breast carcinoma; a primary human breast carcinoma cell line. The patient provided written consent for the transfer of biological material.
The cell lines were cultured in RPMI-1640 nutri- ent medium with the addition of fetal bovine serum (FBS), in a CO2 incubator at a temperature of 37 �C and 5 % carbon dioxide content. To obtain the pri- mary line, the tumor fragment obtained from the patient was placed in a nutrient medium with the addition of gentamicin (10 %), after which it was treated with ethyl alcohol (70 %). The fragment was crushed, centrifuged for 2 minutes at 3000 rpm, and then treated with collagenase solution. The resulting suspension was cultured in a CO2 incubator, after which the cells were filtered and centrifuged for 2 minutes at 3000 rpm. Next, the suspension was washed with sterile DMEM with 5 % FBS, transferred to a T25 vial in 5 ml of DMEM
+ 10 % FBS medium. and the cells were cultured using the method described above. To obtain a suspension of cells from xenograft, a fragment of tumor tissue obtained from the patient was washed in a nutrient medium with an antibiotic (gentamicin), cleaned of necrosis fragments, con- nective tissue and blood vessels. After purifica- tion, the tumor fragment was crushed in a tissue disaggregation system using the automated BD Medimachine system (BD, USA) by adding 1 ml of RPMI-1640 nutrient medium. After crushing the tu- mor tissue, the resulting suspension was selected and filtered through nylon filters with a cell diam- eter of 70 microns and the resulting suspension of BC cells was injected into the nutrient medium of experimental animals.
Recipient animals
Female mice of the Balb/c Nude line in the amount of 36 heads, weighing 20-22 grams, aged 4 weeks, were used as recipient animals, purchased at the Scientific Research Institute "Nursery of Laboratory Animals" of the FIBH RAS. The animals were kept in SPF conditions of the testing laboratory center of the NMRC for Oncology, Ministry of Health of the Russian Federation, in individually ventilated cages at a temperature of 21-23 �C, with free access to water and feed. The animals were divided into 6 groups of 6 mice each. In group 1, there were animals with sub- cutaneous injection of a disaggregated tumor frag-
ment; group 2 - subcutaneous injection of VT20 cell culture; group 3 - subcutaneous injection of primary cell culture; group 4 - orthotopic injection of a dis- aggregated tumor fragment; group 5 - orthotopic injection of VT20 cell culture; group 6 - orthotopic injection of primary cell culture.
All manipulations during the experiment were performed in compliance with the ethical principles established by the European Convention for the Pro- tection of Vertebrate Animals Used for Experiments or Other Scientific Purposes (ETSN 123, Strasbourg, March 18, 1986). Study Protocol No. 19/123 dated 08/3/2021 was approved by the local ethics commit- tee of the NMRC for Oncology, Ministry of Health of the Russian Federation.
Development of the BC model
A skin fold was made on the side of the animal to create a subcutaneous tumor model, into which
0.3 ml of cell suspension was injected in RPMI-1640 nutrient medium.
Anesthesia was administered in two-stages to cre- ate an orthotopic model of animals: premedication with xylazine (20 mg/kg) and anesthesia with zoletil (50 mg/kg). Anesthetized animals were injected into the fatty tissue of the mammary gland with a suspen- sion of tumor cells in a volume of 0.2 ml in a nutrient medium RPMI-1640. Each animal was injected with 3*107 cells.
Tumor growth was observed for 28 days from the moment of injection and measurements of tumor nodes were carried out 2 times a week until the end of the experiment. The volume of tumor nodes was calculated by the formula:
V = (L*W*H) / 6*p, where
V is the volume of the tumor node; L is the length of the tumor node; W is the width of the tumor node; H is the height of the tumor node.
After 28 days, the animals were euthanized in a CO2 chamber.
Statistical analysis of the results
The Microsoft 10 and Statistica 10 software pack- ages were used to analyze the results. The Shapiro- Wilk criterion was used to check for the normality of the obtained data sample. The results were evaluat- ed using median and percentiles. The nonparamet- ric Mann-Whitney criterion was used to assess the reliability of the differences.
STUDY RESULTS AND THEIR DISCUSSION
In the course of this work, the dynamics of the growth of tumor nodes at different injection sites was determined. With subcutaneous injection, the most successful was the injection of tumor cells, a certified VT20 line. At the end of the experiment, the medi- an of tumor nodes in this group was 100.32 [91.15; 113.99] mm3. The growth of tumor nodes was noted in 5 animals in the group (83.33 %). In the group with subcutaneous injection of primary tumor cell culture on the 28th day of the experiment, the median of tumor nodes was 88.79 [86.60; 90.86] mm3, which is
11.49 % less than in the group with the introduction of a certified culture. The formation of tumor nodes was observed in 5 animals (83.33 %). The lowest growth dynamics was observed in the group with a disag- gregated tumor, where the median tumor node was
41.28 [32.96; 44.73] mm3, which is 58 % less than in the group with a certified cell culture. Tumor nodes were observed in 4 animals in the group (66.67 %). Data on the growth dynamics of subcutaneous tumor nodes are shown in Figure 1.
During orthotopic injection of tumor materi- al, it was revealed that in this growth variant, the median volume of tumor nodes in the group with certified VT20 cell culture and primary cell culture reached similar values - 149.22 [145.43; 153.58]
and 148.25 [144.09; 149.81] mm3. During this im- plantation, the growth of tumor nodes was observed in all 6 animals in the groups (100 %). In the group with the injection of a disaggregated tumor, the me- dian volume of tumor nodes on the 28th day of the experiment was 73.24 [70.11; 78.19] mm3, and the presence of tumor nodes was observed in 4 animals in the group (66.67 %). Data on the growth dynamics of orthotopic tumor nodes are presented in Figure 2. A comparative analysis of the growth dynamics of tumor nodes in subcutaneous and orthotopic versions by injection of tumor cells revealed that both tumor lines and the resulting suspension of cells reached a significantly larger volume during orthotopic injection. The data on the comparative analysis of the growth of tumor nodes are presented
in Figure 3.
According to the results obtained, it was revealed that orthotopic injection of tumor cells showed a significantly higher growth rate than subcutane- ous injection. As well as analyzing various literature
data, we confirmed that orthotopic implantation is recommended for faster growth and achieving a larger volume of the tumor node [11, 12]. When assessing the growth characteristics of xenografts, an analysis of the growth of human cardioesopha- geal cancer in mouse models was carried out in one of the works of the NMRC for Oncology, Ministry of Health of Russia [12]. In the work of Kit S. O. et al. (2020), as well as in our work, a significant influ- ence of the implantation site on the growth dynam- ics of the tumor node and the chance of xenograft engraftment was revealed, which is probably due to the influence of the environment surrounding the tumor node [12]. In the studies of Zibirov R. F. and Moserov S. A. (2018), Chen S. et al. (2023), it was shown that the tumor microenvironment through signaling molecules contributes to the successful engraftment of the tumor fragment, the growth of the tumor node, the initiation of neovasculation and the formation of metastases [13, 14]. This microen- vironment is represented by a stroma with cells of various types, such cells include tumor-associated fibroblasts - in Zhang Ya's et al. study (2023), it was shown that these cells are activated by microenvi- ronment factors such as TGF-�, monocytic chemo- tactic protein -1, fibroblast growth factor; they pro-
duce signaling proteins such as hepatocyte growth factor, insulin-like growth factor-1, stimulating the proliferation of tumor cells [15]. In addition, in the studies of Pastushenko E. with co-authors (2018) and Kuburich N. A. et al. (2023), the effect of tumor- associated fibroblasts on the induction of epithelial- mesenchymal transition (EMF) was demonstrated by the secretion of TGF-�, which activates genes encoding proteins necessary for mesenchymal cell functions (vimentin, N-cadherin, fibronectin-1) and suppressing the expression of proteins important for the epithelial phenotype (E-cadherin, cytokeratins and lamins) [16, 17]. The tumor microenvironment also includes T and B lymphocytes: in the work of Zibirov R. F. and Moserov S. A. (2018), a high content of interleukin-10 (IL-10), produced by tumor cells and contributing to the inhibition of cytotoxic activity of T lymphocytes, which contributes to the survival of tumor cells in the body, was revealed [13]. Data on the effect of B lymphocytes on tumor pathogenesis are ambiguous - in the work of Qin Yu. et al. (2021), it was shown that tumor infiltration by B lympho- cytes is a positive prognostic marker. Such cells per- form an antigen-presenting function and express CD80, CD86 molecules, activating CD4+ and CD8+ T cells [18]. However, in a study by Lindner S. et al.
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Fig. 1. Dynamics of growth of subcutaneous BC tumor nodes in the group with a disaggregated tumor, with injection of VT20, with injection of primary culture
Note: The data is presented as a median
Fig. 2. Growth dynamics of orthotopic tumor nodes BC in the group with a disaggregated tumor, with injection of VT20, with injection of primary culture
Note: The data is presented as a median
(2013), it was shown that regulatory B cells secrete interleukin-10, interleukin-35, interleukin-6, TGF-�, contributing to the immunosuppression of the anti- tumor reaction [19]. Also, one of the main cells of the tumor microenvironment are mast cells that activate angiogenesis through histamine, heparin, the main fibroblast growth factor, vascular endothelial growth
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factor, TGF-� [13]. In the work of Liu S. et al. (2023), despite various contradictory data, the protumoro- genic effect of mast cells in malignant formations of various diseases was shown [20]. Thus, orthotopic implantation of tumor cells into the body of an ex- perimental animal contributes to the development of an appropriate microenvironment response, which, according to the analyzed literature and experimental data, contributes to more successful engraftment and growth of xenograft.
In our work, we also analyzed the effect of the type of transferred material on its survival and growth in the recipient's body. In the course of our work, we noted the most active growth and greater success of graft engraftment of the certified BT 20 cell line, however, the primary cell line formed in our institu- tion also demonstrated a result close to the certified culture. The disaggregated tumor obtained from the patient had the lowest growth dynamics and the per- centage of engraftment. Analyzing the literature data on the topic, we noted the need for intercellular com- munication for the development of physiological and pathological processes [21]. In laboratory practice, there are several ways to obtain a suspension of tu- mor cells: enzymatic, chemical and mechanical [22]. Proteolytic enzymes such as papain, trypsin, prote-
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Fig. 3. Comparison of the growth dynamics between the groups of a disaggregated tumor, with VT20 injection, with primary culture injection with subcutaneous and orthotopic injection
Note: the data are presented as a median, * - statistically significant differences between the groups according to the Mann-Whitney criterion (p < 0.05)
ase, elastase and hyaluronidase are often used for enzymatic dissociation [23]. In a study by Janek K. et al. (2016), an enzymatic mixture was used to ob- tain a suspension of B C tumor cells, which included collagenase, a solution of dyspase and DNase [24]. However, according to a study by Nishikant T. and co-authors (2013), the most effective method of en- zymatic dissociation against a breast tumor was the use of dispase II [25]. During chemical dissociation, it is necessary to achieve the leaching of calcium and magnesium cations from cells, in view of their important role in maintaining the integrity of the cell surface [26]. In a study by Damm G. et al. (2019), EDTA was used for chemical dissociation, which promotes the removal of Ca2+ and Mg2+ cations and leads to a decrease in intercellular interactions. Their work also describes the use of hypertonic solutions of sucrose, maltose and lactose to disrupt the gap contacts between cells [27]. Mechanical dissociation of tumor tissue is a simple and effective method of obtaining a cell suspension, consisting in crushing the resulting sample with scissors, homogenizing and filtering the resulting suspension [23]. Thus, in the work of Krbala L. et al. (2017), using a mechan- ical dissociation method, it was possible to form a primary cell line of human colorectal cancer ob- tained from a primary tumor with an efficiency of
39.4 %, and a cell line isolated from the correspond-
ing metastases in the lymph nodes had an efficiency of up to 70 % [22]. However, many researchers be- lieve that mechanical dissociation is more traumatic
for cells than other methods and leads to significant cell death, which is not suitable for obtaining tumor cells [28-30].
Based on various literature data, it can be as- sumed that the use of enzymatic or chemical dis- sociation methods in our work with respect to the primary tumor could contribute to more successful engraftment of samples and greater growth dynam- ics of the obtained xenografts than with mechanical grinding of the sample. Determining an effective way to develop a human BC tumor model is necessary for us to conduct further studies of the nature of the course of this disease, as well as evaluate the effectiveness of new treatment methods.
CONCLUSION
In the course of the work, the growth dynamics of orthotopic and heterotopic in vivo models of breast cancer were evaluated. With orthotopic injection of tumor material, a higher percentage of engraftabil- ity was observed (66.67 %; 100 %). In addition, the primary BC line obtained in the course of this work had a growth dynamics of tumor nodes close to the certified culture, which gives grounds to use this line in further studies. In conclusion, it can be noted that we have developed an adequate BC tumor model for various methods of implantation of the material and with the possibility of further use in the study of mechanisms of carcinogenesis and testing of new pharmacological substances.
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Information about authors:
Inna S. Lyashenko - PhD student, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation SPIN: 5074-2896, AuthorID: 1165761
Maria V. Romanova - junior researcher at testing Laboratory Center, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0001-8734-9210, SPIN: 5148-0830, AuthorID: 1032029, Scopus Author ID: 57217235360
Anna S. Goncharova 12 - Cand. Sci. (Biol.), head of testing Laboratory Center, National Medical Research Centre for Oncology, Rostov-on-Don,
Russian Federation
ORCID: https://orcid.org/0000-0003-0676-0871, SPIN: 7512-2039, AuthorID: 553424, Scopus Author ID: 57215862139
Daria V. Khodakova - junior researcher at testing Laboratory Center, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0003-3753-4463, SPIN: 8718-3983, AuthorID: 1056414, Scopus Author ID: 57221463056
Anastasia V. Galina - junior researcher at testing Laboratory Center, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0001-7823-3865, SPIN: 9171-4476, AuthorID: 1071933, Scopus Author ID: 57221460594
Sofya V. Gurova - junior researcher at testing Laboratory Center, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0002-9747-8515, SPIN: 5413-6901, AuthorID: 1147419
Svetalna Yu. Filippova - researcher, laboratory of cell technologies, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0002-4558-5896, SPIN: 9586-2785, AuthorID: 878784, Scopus Author ID: 57189618843
Yuliana S. Shatova - Dr. Sci. (Med.), leading researcher, Department of Soft Tissue and Bone Tumors, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation
SPIN: 8503-3573, AuthorID: 294376
Contribution of the authors:
Lyashenko I. S. - conducting an experiment; Romanova M. V. - writing the source text;
Goncharova A. S - formulation of the research concept; Khodakova D. V. - search of literary sources;
Galina A. V. - analysis of the obtained experimental data; Gurova S. V. - final conclusions;
Filippova S. Yu. - conducting an experiment; Shatova Yu. S. - scientific management.
