Investigation of Combined Effects of KRAS-G12V and KRAS- 2 LCS6 (rs61764370) Alterations on Tumor Progression and Me- 3 tastasis in HNSCC by Using Xenograft Mouse Model

Investigation of Combined Effects of KRAS-G12V and KRAS- 2 LCS6 (rs61764370) Alterations on Tumor Progression and Me- 3 tastasis in HNSCC by Using Xenograft Mouse Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of Combined Effects of KRAS-G12V and KRAS- 2 LCS6 (rs61764370) Alterations on Tumor Progression and Me- 3 tastasis in HNSCC by Using Xenograft Mouse Model M. Burak Kaplan, Vildan Betul Yenigun, Yasemin Sena Acar, Hatice Toy, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7398588/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Head and neck cancers (HNCs) are the seventh most common cancers worldwide, with KRAS mutations playing a key role for developing and progression of cancer. The G12V mutation is one of the most commonly identified KRAS mutation and has been associated with tumor progression and aggressive disease. Additionally, T > G alteration in the binding site of miRNA let-7 of the KRAS -3'UTR (rs61764370), let-7 complementary site 6 (LCS6), has been found to promote cancer development, metastasis, and treatment resistance. Material and Methods HEp2 laryngeal cancer cells, transfected with plasmid constructs carrying the KRAS -G12V mutation and the combined G12V + LCS6 mutations were transplanted subcutaneously (SC) or intraperitoneally (IP) into CD-1 nude mice with non-transfected control cells. The mice were euthanized on the 40th day post-transplantation and examined histopathologically. Results Primary tumor development was observed in all cell-transplanted mice. The G12V mutation significantly promoted tumor growth (p < 0.01), and LCS6 alteration further accelerated progression (p < 0.001). No metastases occurred in SC-injected mice, but IP-injected mice with KRAS mutations developed metastases. G12V alone led to only liver metastases (50%), whereas G12V + LCS6 resulted in metastases in the liver (100%), lung (75%), kidney (25%), and paraaortic lymph nodes (75%). No brain metastases were observed in any group. Conclusion This study shows that while the KRAS -G12V mutation enhances HNSCC tumor growth and metastasis at relatively low rates, its combination with the LCS6 alteration significantly accelerates both. These findings suggest that miRNAs and miRNAs binding site mutations in KRAS -3’UTR contribute to cancer aggressiveness and could serve as potential therapeutic targets. KRAS G12V rs61764370 xenograft mouse model metastasis HNSCC Figures Figure 1 Figure 2 1. Introduction Head and neck cancers (HNCs), over 90% of which are classified as Head and Neck Squamous Cell Carcinoma (HNSCC), include malignancies affecting the lip, oral cavity, nasopharynx, oropharynx, hypopharynx and larynx regions [ 1 ]. HNCs rank as the seventh most common type of cancer worldwide [ 1 ]. In terms of mortality, HNCs account for 6.3% of all cancer-related deaths globally [ 2 ]. Given the rapid progression of HNSCC, many patients are diagnosed at advanced stages with metastasis [ 3 ]. The presence of metastasis is associated with poor prognosis and high mortality rates in HNSCC patients [ 4 , 5 ]. While the 5-year survival rate for all HNSCC patients in the USA is 68.5%, this rate drops significantly to 39.3% in those with metastatic disease [ 6 ]. Among these, distant metastasis is particularly considered one of the most important factors in terms of disease recurrence and mortality [ 7 ]. Although significant progress has been made in understanding the disease mechanisms, the genetic and epigenetic factors driving the progression of the disease are still not fully elucidated [ 8 ]. Unlike the stochastic model, which suggests that all tumor cells can contribute to cancer growth and progression, the cancer stem cell (CSC) hypothesis proposes that only a specific subpopulation of cells within the tumor is responsible for tumor initiation, growth and spread [ 9 , 10 ]. CSCs have also been associated with the development of metastases and increased mortality rates [ 11 , 12 ]. The KRAS gene is a key proto-oncogene frequently mutated in various cancers [ 13 ]. Particularly, the G12V mutation is one of the most prevalent KRAS mutations and has predictive importance in many cancer types [ 14 ]. Beyond coding regions, 3’ untranslated region (3’UTR) mutations of the KRAS gene are also known to have clinical significance [ 15 – 17 ]. Since the 3’UTR regions of genes are the target sequences of microRNAs (miRNAs) [ 18 ], mutations in these regions disrupt the miRNA binding and trigger tumorigenesis. Among these binding sites, it has been demonstrated that the T > G nucleotide change in the LCS6 site (rs61764370) attenuates miRNA let-7 binding, resulting in increased KRAS expression, prolonged KRAS mRNA intracellular residence time and reduced let-7 levels [ 19 , 20 ]. Increased KRAS expression has also been associated with a more aggressive disease course in laryngeal cancer [ 21 ]. Xenograft mouse models are widely used to investigate the mechanisms underlying tumor development and metastasis providing more valuable data compared to in vitro cell models [ 22 ]. However, so far, there is no any study investigating the combined effects of these two KRAS alterations (G12V and rs61764370) on tumor progression and metastasis, and no in vivo studies have been conducted to date. In the present study, we aimed to investigate the simultaneous effects of KRAS -G12V mutation and LCS6 alteration in KRAS -3'UTR region on the progression and metastasis of HNSCC by using xenograft mouse model and also to evaluate associated histopathologic data. 2. Materials and Methods 2.1. Cell Culture The metastatic laryngeal cancer cell line HEp-2 (ATCC® CCL-23, USA) was used for transfection and tumor formation in the xenograft mouse model. HEp-2 cells were cultured in RPMI 1640 medium (Biochrome, Germany) supplemented with 10% fetal bovine serum (FBS, Biochrome, Germany) and 1% penicillin/streptomycin (P/S; 100 U/ml penicillin and 100 µg/ml streptomycin, Biochrome, Germany). The cells were maintained in an incubator at 37°C with 5% CO₂. 2.2. KRAS Plasmid Constructs and Transfection to HEp-2 Cells As described in our previous study [ 23 ], plasmid constructs carrying KRAS -G12V mutation with and without LCS6 alteration were generated. Plasmid constructs were generated, containing a G12V mutant coding sequence (CDS) region and a normal LCS6 region of the KRAS gene ( KRAS -CDSm-LCS6n) and a G12V mutant CDS region and a mutant LCS6 region of the KRAS gene ( KRAS -CDSm-LCS6m) were constructed using the pLenti-CMV-GFP-2A-Puro (8950 bp) bi-cistronic lentiviral vector (Applied Biological Materials Inc., Canada). The constructs, including the empty plasmid, were transfected into HEp-2 cells using the amphotropic retroviral packaging cell line 2A (ATCC® CRL 12013, USA). For this purpose, 2A cells were transfected with a mixture of the constructs and Lipofectamine® 2000 (Invitrogen, USA). The constructs were subsequently packaged into viral particles and transferred to the cancer cells. Transfected HEp-2 cells were treated with 0.2 µg/ml puromycin to eliminate non-transfected cells, achieving a transfection efficiency of approximately 80–90% after one month. To simplify the tracking of different HEp-2 cell types containing plasmid constructs in the experimental systems, the following abbreviations were assigned: HEp2-0: Cells without plasmid transfection, HEp2-1: Cells containing pLenti- KRAS -CDSm-LCS6m plasmid construct, HEp2-2: Cells containing pLenti- KRAS -CDSm-LCS6n plasmid construct, HEp2-3: Cells containing pLenti-CMV-GFP-2A empty plasmid vector. 2.3. Isolation of Cancer Stem Cell (CSC) Stem cells were also isolated to generate xenograft mouse models expressing pLenti- KRAS -CDSm-LCS6n and pLenti- KRAS -CDSm-LCS6m. Control cells, along with cells containing plasmid constructs and empty plasmids, were trypsinized (Biochrome, Germany) and washed with 1x phosphate-buffered saline (PBS) (Biochrome, Germany) prior to antibody labeling. The cells were then incubated with CD44 (APC-conjugated) and CD24 (PE-conjugated) antibodies (Thermo Scientific, USA) in 100 µl of 2% fetal bovine serum (FBS) in Hank's Balanced Salt Solution (HBSS). Incubation was carried out for 1 hour at 4°C in the dark, with gentle agitation every 10 minutes. The cells were washed twice; first with 3 ml of 2% FBS/HBSS to remove unbound antibodies, and then with HBSS alone. The final cell pellet was resuspended in 400 µl of 2% FBS/HBSS. Cell sorting was performed by using a FACS Aria III device (BD Pharmingen, USA) to isolate the desired populations. 2.4. Preparation of Cells for Xenograft Model To create a xenograft model, both total cells and stem cells were transferred to mouse groups. HEp2-0, HEp2-1, HEp2-2, and HEp2-3 cells were detached from the plates using Trypsin/EDTA (0.25%, Biochrome, Germany) and washed with the complete medium. All cell groups, including isolated stem cell groups, were counted using trypan blue dye (Gibco, USA). 9.5x106 cells for total cells (TCs) and 3.2x105 cells for isolated stem cells (CSCs) were counted and suspended in 200 µl medium for each group. Then, 50 µl matrigel (STEMCELL Technologies, Canada) was added to each cell group on the ice. 2.5. Xenograft Mouse Experiments In the present study, 4-6-week-old CD1 nude male mice were used. The study was approved by Selcuk University Animal Experiments Local Ethics Committee (2015-00015). Mice were obtained from Kobay Experimental Animals Laboratory Inc. (Ankara, Turkiye) and Yıldırım Beyazıt University Experimental Application and Research Center (Ankara, Turkiye). The experiments were carried out in Individually Ventilated Cages (IVC) specifically designed for CD1 nude mice at Selcuk University Experimental Medicine Research and Application Center. All procedures were performed in accordance with humane and ethical treatment protocols to ensure comfort and minimal pain. All mice were housed at 22 ± 2°C, 50% humidity and a 12-hour light-dark cycle. Throughout the study, mice had ad libitum access to sterilized or disinfected tap-water and standard foot pellets. Bedding and litter trays were changed twice a week. The mice were kept under the aforementioned conditions for 2 weeks and the experiments began with animals weighing 18–20 g. The animals were initially divided into two groups according to tumor injection sites: intraperitoneal (IP) and subcutaneous (SC) using a 26-gauge needle (Beybi, Turkiye) under sterile conditions. Each group was then further subdivided into four subgroups based on the transferred cell types (HEp2-0, HEp2-1, HEp2-2, HEp2-3). Additionally, one group was designated as a control without any procedure. 2.6. Tumor Monitoring In the groups treated with cells via the SC route, all tumor progression was monitored by inspection and palpation of the application site twice a week. In contrast, since palpation was not feasible, the groups receiving cells by the IP route were evaluated by observing the general health status and changes in body weight of the mice. The body weight of all mice and the tumor masses (width x length) in the SC-transplanted mice were systematically measured and recorded. These measurements were performed twice a week by the same investigator. On 40th day, determined by the general condition of the mice, the mice were sacrificed in the CO2 chamber. Tumor tissues and the other organs were collected for histopathological examination. 2.7. Histopathological Analysis For histopathological examination, tumor tissues and other organs were fixed in 10% buffered formalin overnight at 4°C and embedded in paraffin. Paraffin blocks were sectioned at a thickness of 3–4 microns using a microtome, and slides were prepared. After deparaffinization, slides were stained with Hematoxylin and Eosin (H&E) and examined under a bright-field microscope (Nikon, Japan). 2.8. Statistical Analysis All statistical analyses of the data obtained were performed with GraphPad Prism® V.10.4.1 software (GraphPad Software Inc., USA). Student's t-test and one-way ANOVA were used for group comparisons. A p-value < 0.05 was considered statistically significant. 3. Results 3.1. Transfection of plasmid constructs into the cells As demonstrated in our previous study [ 23 ], two methods were utilized to confirm the successful transfection of plasmid constructs into the cells. First, the transfected cells were observed under a fluorescence microscope, as the plasmids contained a green fluorescent protein (GFP) marker. Second, the expression of non-mutant and mutant KRAS (G12V-specific) proteins were evaluated via western blot analysis. The results revealed significantly higher levels of mutant KRAS protein in HEp2-1 and HEp2-2 cells compared to HEp2-0 and HEp2-3. These findings validate the successful transfection of G12V mutant KRAS plasmid constructs into HEp2 cells. 3.2. Tumor Formation and Monitoring For the optimization study, the mice were divided into groups as described above, and either TCs or CSCs were transplanted into separate groups of mice. Both cell groups were administered via SC or IP routes to different groups of mice. While 9.5x10 6 cells were used for the TC injection, 3.2x10 5 cells were used for the CSC injection. In all cell-transplanted mice, tumor formation was observed; however, there was no significant difference (p > 0.05) in tumor size between TC- and CSC-transplanted mice. In terms of tumor development, the effect of approximately 3×10⁵ HEp2 CSCs was found to be equivalent to the effect of 10×10⁶ HEp2 TCs. Therefore, the subsequent experiments were performed using mice transplanted with TCs. In each group transplanted with cells, weight gain was observed until day 30–33, followed by a significant weight loss from day 34–38 (Fig. 1a). On the 3rd day after cell transplantation, masses were observed at the injection sites in all mice that received tumor cells via the SC route (Fig. 1b). However, because the masses had not yet reached a measurable palpable hardness, measurements of the masses were initiated on day 14. Our results showed that the G12V mutation increases the growth rate of the primary tumor, and the presence of the LCS6 alteration further accelerates tumor growth (Fig. 1c). On the 40th day, the weights of the primary tumor masses excised from the sacrificed mice were measured (Figs. 1d, 1e, 1f). Accordingly, when all cell types were evaluated together, the primary tumor masses grew faster in SC-transplanted mice compared to IP-transplanted mice. The mice were dissected, and the abdomen, thoracic cavity and skull were opened for macroscopic examination. No macroscopic metastasis was observed in any of the SC-transplanted mice. IP-transplanted mice, metastases were observed in mice injected with HEp2-1 and HEp2-2 cells, whereas no macroscopic metastases were detected in mice injected with HEp2-0 or HEp2-3 cells. In the group injected with HEp2-1 cells, tumor development showed a greater spread within the abdominal region compared to HEp2-2 (Figs. 1g, 1h). This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. 3.3. Histopathological assessment After dissection, the primary tumor tissues, potential metastatic sites, and suspected tissues were histopathologically examined. No metastasis was detected in mice transplanted with HEp2-0 and HEp2-3 cells, whereas metastases were observed in cells carrying KRAS -G12V mutation. Among these, only liver metastasis (50% of cases) was detected in mice transfected with HEp2-2 cells carrying only the G12V mutation, whereas in mice transfected with HEp2-1 cells carrying also the G12V + LCS6 alteration, lung (75%), kidney (25%) and regional lymph node (75%) metastases were detected in addition to liver metastases. It was also observed that LCS6 alteration increased the frequency of liver metastases (100%). No brain metastases were observed in any cell group (Table 1 , Fig. 2a-e). Table 1 Metastatic status in mice with tumor development. HEp2 Cells Number of Mice with Metastases / Tumors Mouse No Organ/Tissue Metastasis Condition Primary Tumor Lung Liver Kidney Brain Regional Lymph Node HEp2-0 1 + - - - - - 0/3 2 + - - - - - 3 + - - - - - HEp2-1 4/4 1 2 + + - + + + - - - - + - 3 4 + + + + + + + - - - + + HEp2-2 2/4 1 2 + + - - + - - - - - - - 3 4 + + - - + - - - - - - - HEp2-3 1 + - - - - - 0/3 2 3 + + - - - - - - - - - - 4. Discussion Genetic alterations provide significant insights into the etiology and clinical progression of cancer. In particular, nucleotide changes in proto-oncogenes play an increasingly critical role in cancer diagnosis and treatment. One of the most important proto-oncogenes, the KRAS gene, is notable not only for mutations within its coding region but also for alterations in its 3’UTR. These alterations possess substantial clinical relevance in the field of oncology [ 15 ]. In cancer research, the xenograft mouse model is a widely utilized method to examine the growth and metastatic behavior of cancer cells in a living biological system. In the literature, both TC and CSC applications are employed to develop xenograft mouse models. While the CSC xenograft model is used for studying the hematopoietic system and certain cancers [ 24 , 25 ], the TC model is frequently utilized in laryngeal carcinoma research [ 26 , 27 ]. In our study, both cell types were used, and tumor development was observed in both models. This finding aligns with the stochastic model that posits that all tumor cells can contribute to cancer development. In the literature, the number of injected cells for TC applications ranges between 1–10×10⁶, while for CSC applications, 1–5×10⁵ cells are sufficient for cancer development [ 28 ]. These values are consistent with the cell counts used for tumor formation in our study. Given that the mass is not detectable by palpation in IP-transplanted mice, the SC transplantation method is frequently used in the literature for tumor size monitoring. Our finding that the IP method is more effective for metastasis tracking aligns with previous studies. In their xenograft mouse model study using a colorectal cancer cell line, Taibi, et al. [ 29 ] observed no metastasis in mice that received SC injections, whereas metastases were detected in those that underwent IP injections. Similarly, in our study, no metastases were observed in mice that received SC injections, whereas both macroscopic and histopathological metastases were detected in mice that underwent IP injections. This finding is consistent with the existing literature. In the literature, the sacrifice timing in xenograft mouse model studies varies depending on the type of cancer [ 30 ]. For aggressive cancers, this period can be as short as 14–21 days [ 31 ], whereas in less aggressive, non-metastatic cancers or in mice receiving hematopoietic cell transplants, it can extend up to six months [ 32 ]. In our study, the optimal dissection time for HNSCC was determined to be day 40, based on the cessation of weight gain and the onset of weight loss in mice. In the literature, tumor formation at the injection site can be observed within the first week in xenograft mouse models of cancer [ 33 ]. Similarly, in our study, the development of tumors was detected in all mice by day 3 following SC cell transplantation. The KRAS -G12V mutation has been known to play a pivotal role in the development of cancer [ 34 ]. In the present study, it was observed that this specific mutation led to a substantial augmentation in the primary tumor's growth rate. The other KRAS alteration analyzed in our study, the T > G substitution in 3'UTR-LCS6 (rs61764370), has been linked to multiple cancers, including ovarian cancer [ 35 , 36 ], breast cancer [ 17 , 20 , 37 – 39 ], lung cancer [ 40 , 41 ], colorectal cancer [ 16 ] and HNCs [ 15 , 42 ]. However, the impact of this variation differs across various cancer types. Crowley, et al. [ 43 ] investigated the rs61764370 variant in the SW48 colorectal cancer cell line and found that while it did not affect KRAS expression, it reduced let-7 levels and increased cell proliferation [ 43 ]. In contrast, Hollestelle, et al. [ 44 ] reported no significant effect of this variant in ovarian and breast cancers [ 44 ]. In our study, we observed that while the G12V mutation alone promoted tumor growth in laryngeal cancer, the co-occurrence of the G12V mutation with the LCS6 variant further enhanced tumor growth (p < 0.001). The KRAS -LCS6 (rs61764370) variant is known to play a role in cancer progression; however, there are only a limited number of studies investigating its impact on metastasis. While previous observational studies have suggested that this variant increases metastatic potential in colorectal cancer, osteosarcoma, and gallbladder cancer [ 16 , 45 , 46 ], no significant in vivo studies have been conducted on this subject. In our study, we examined the in vivo effects of both the G12V and LCS6 mutations. Our findings indicate that in the presence of the G12V mutation alone, metastasis was observed only in the liver (50% of cases). However, when both the G12V and LCS6 mutations were present, metastases were detected in the liver (100%), lungs (75%), kidneys (25%), and regional lymph nodes (75%). Notably, no brain metastases were observed in any group. Given that HNSCC predominantly spreads locally, brain metastases are rare [ 47 ]. This characteristic likely explains the absence of brain metastases in any of the mice in our present study. Ulusan, et al. [ 48 ] reported that the rs61764370 variant is associated with an increased risk of lymph node metastasis in HNCs. Similarly, in our study, the group carrying the LCS6 variant exhibited a high incidence of lymph node metastasis, providing in vivo validation for the clinical observation. Furthermore, in our previous in vitro cell model study reported that the LCS6 variant had no additional effect on tumor growth or invasion capacity beyond the G12V mutation [ 23 ]. However, our in vivo mouse model study demonstrated a significant difference, highlighting the importance of complementing in vitro studies with in vivo research in cancer investigations. 5. Conclusions This study demonstrates that, in xenograft mouse model, the KRAS -G12V mutation increases HNSCC tumor growth and metastasis at relatively low rates, whereas in the combination with the LCS6 alteration, both tumor growth and metastasis are significantly increased. The LCS6 alteration increases specifically metastasis to the liver, lungs, kidneys, and regional lymph nodes. In conclusion, our findings make a significant contribution to the literature on the impact of KRAS -G12V and LCS6 alterations on cancer progression and metastasis. They may also serve as a guide for future studies aimed at developing new therapeutic strategies targeting these alterations. Furthermore, the difference between our in vitro and in vivo results highlights the critical importance of in vivo models in cancer research to better understand the progression of tumors and clinical outcomes. Abbreviations The following abbreviations are used in this manuscript: HNCs Head and neck cancers HNSCC Head and neck squamous cell carcinoma CSC Cancer stem cell TC Total cell 3’UTR 3’ untranslated region LCS6 Let-7 complementary site 6 CDS Coding sequence IP Intraperitoneal SC Subcutaneous GFP Green fluorescent protein Declarations Author Contributions: Conceptualization, H.A.; methodology, H.A., H.T. and K.O.; validation, H.A., M.B.K., V.B.Y. and Y.S.A.; formal analysis, H.A., M.B.K. and Y.S.A.; investigation, M.B.K., V.B.Y., Y.S.A., H.T. and K.O.; resources, H.A., M.B.K., H.T. and K.O.; data curation, H.A.; writing—original draft preparation, M.B.K.; writing—review and editing, H.A. and V.B.Y.; visualization, M.B.K., H.T. and K.O.; supervision, H.A.; project administration, H.A.; funding acquisition, H.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially funded by the Scientific and Technological Research Council of Türkiye (TÜBİTAK), grant number 112S498. Institutional Review Board Statement: The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Selcuk University Animal Experiments Local Ethics Committee (2015-00015 and 10.01.2015). Data Availability Statement: The data supporting this study's findings are available from the corresponding author upon reasonable request. Acknowledgments: We thank Professor Thomas E. Carey, University of Michigan, USA, for supplying the cell line. Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. 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Journal of Cancer Research and Clinical Oncology 2016 , 142 , 2577-2583, doi:10.1007/s00432-016-2254-9. Djalilian, H.R.; Tekin, M.; Hall, W.A.; Adams, G.L. Metastatic head and neck squamous cell carcinoma to the brain. Auris Nasus Larynx 2002 , 29 , 47-54, doi:https://doi.org/10.1016/S0385-8146(01)00113-4. Ulusan, M.; Sen, S.; Yilmazer, R.; Dalay, N.; Demokan, S. The let-7 microRNA binding site variant in KRAS as a predictive biomarker for head and neck cancer patients with lymph node metastasis. Pathology-Research and Practice 2022 , 239 , 154147. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":801944,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) The change in the average weight of transplanted mice until dissection. (\u003cstrong\u003eb\u003c/strong\u003e) Images of masses developed in SC-transplanted mice on days 3 (left) and 10 (right). (\u003cstrong\u003ec\u003c/strong\u003e) Graph of tumor growth over time in SC-transplanted mice. (\u003cstrong\u003ed\u003c/strong\u003e) A sacrificed mouse (left) and macroscopic appearance of the SC primary tumor (right). (\u003cstrong\u003ee\u003c/strong\u003e) Primary tumor masses in SC-transplanted mice with HEp2-1 (left) and HEp2-2 (right) cells. (\u003cstrong\u003ef\u003c/strong\u003e) Graph showing the average weights of primary tumor masses excised after dissection (*=p\u0026lt;0.05, **=p\u0026lt;0.01, ***=p\u0026lt;0.001, and ****=p\u0026lt;0.0001). (\u003cstrong\u003eg\u003c/strong\u003e) Macroscopic view of the abdomen and thoracic cavity in IP-transplanted mice with HEp2-1 (left) and HEp2-2 (right). (\u003cstrong\u003eh\u003c/strong\u003e) Macroscopic images of the liver in IP-transplanted mice with HEp2-1 (left) and HEp2-2 (right).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7398588/v1/10eaf29b9614aec450751146.png"},{"id":94399046,"identity":"5e043629-cd19-432b-a0d2-f4713cbfa8f2","added_by":"auto","created_at":"2025-10-27 13:57:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":677652,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological examination. (\u003cstrong\u003ea\u003c/strong\u003e) Histopathology of primary tumors in SC- (left) and IP-transplanted mice (right). (\u003cstrong\u003eb\u003c/strong\u003e) Liver histopathology of mice transplanted with HEp2-0 (left, tumor-negative) and HEp2-1 cells (right, tumor-positive). (\u003cstrong\u003ec\u003c/strong\u003e) Lung histopathology of mice transplanted with HEp2-1 (left, tumor-positive) and HEp2-2 cells (right, tumor-negative). (\u003cstrong\u003ed\u003c/strong\u003e) Kidney histopathology of mice transplanted with HEp2-1 (left, tumor-positive) and HEp2-2 cells (right, tumor-negative). (\u003cstrong\u003ee\u003c/strong\u003e) Histopathology of the regional lymph node from a mouse transplanted with HEp2-1 cells (tumor-positive).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7398588/v1/bdf870fbbc9351bc018ef63e.png"},{"id":95074697,"identity":"86cb3d6e-0949-4c68-9944-54f02d45b20d","added_by":"auto","created_at":"2025-11-04 04:23:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2749376,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7398588/v1/35b1f0e4-a7f6-4da1-8596-eeaaeeac3472.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eInvestigation of Combined Effects of KRAS-G12V and KRAS- 2 LCS6 (rs61764370) Alterations on Tumor Progression and Me- 3 tastasis in HNSCC by Using Xenograft Mouse Model \u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHead and neck cancers (HNCs), over 90% of which are classified as Head and Neck Squamous Cell Carcinoma (HNSCC), include malignancies affecting the lip, oral cavity, nasopharynx, oropharynx, hypopharynx and larynx regions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. HNCs rank as the seventh most common type of cancer worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In terms of mortality, HNCs account for 6.3% of all cancer-related deaths globally [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Given the rapid progression of HNSCC, many patients are diagnosed at advanced stages with metastasis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The presence of metastasis is associated with poor prognosis and high mortality rates in HNSCC patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. While the 5-year survival rate for all HNSCC patients in the USA is 68.5%, this rate drops significantly to 39.3% in those with metastatic disease [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among these, distant metastasis is particularly considered one of the most important factors in terms of disease recurrence and mortality [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although significant progress has been made in understanding the disease mechanisms, the genetic and epigenetic factors driving the progression of the disease are still not fully elucidated [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUnlike the stochastic model, which suggests that all tumor cells can contribute to cancer growth and progression, the cancer stem cell (CSC) hypothesis proposes that only a specific subpopulation of cells within the tumor is responsible for tumor initiation, growth and spread [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CSCs have also been associated with the development of metastases and increased mortality rates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eKRAS\u003c/em\u003e gene is a key proto-oncogene frequently mutated in various cancers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Particularly, the G12V mutation is one of the most prevalent \u003cem\u003eKRAS\u003c/em\u003e mutations and has predictive importance in many cancer types [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Beyond coding regions, 3\u0026rsquo; untranslated region (3\u0026rsquo;UTR) mutations of the \u003cem\u003eKRAS\u003c/em\u003e gene are also known to have clinical significance [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Since the 3\u0026rsquo;UTR regions of genes are the target sequences of microRNAs (miRNAs) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], mutations in these regions disrupt the miRNA binding and trigger tumorigenesis. Among these binding sites, it has been demonstrated that the T\u0026thinsp;\u0026gt;\u0026thinsp;G nucleotide change in the LCS6 site (rs61764370) attenuates miRNA let-7 binding, resulting in increased \u003cem\u003eKRAS\u003c/em\u003e expression, prolonged \u003cem\u003eKRAS\u003c/em\u003e mRNA intracellular residence time and reduced let-7 levels [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Increased \u003cem\u003eKRAS\u003c/em\u003e expression has also been associated with a more aggressive disease course in laryngeal cancer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eXenograft mouse models are widely used to investigate the mechanisms underlying tumor development and metastasis providing more valuable data compared to \u003cem\u003ein vitro\u003c/em\u003e cell models [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, so far, there is no any study investigating the combined effects of these two \u003cem\u003eKRAS\u003c/em\u003e alterations (G12V and rs61764370) on tumor progression and metastasis, and no \u003cem\u003ein vivo\u003c/em\u003e studies have been conducted to date. In the present study, we aimed to investigate the simultaneous effects of \u003cem\u003eKRAS\u003c/em\u003e-G12V mutation and LCS6 alteration in \u003cem\u003eKRAS\u003c/em\u003e-3'UTR region on the progression and metastasis of HNSCC by using xenograft mouse model and also to evaluate associated histopathologic data.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cell Culture\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe metastatic laryngeal cancer cell line HEp-2 (ATCC\u0026reg; CCL-23, USA) was used for transfection and tumor formation in the xenograft mouse model. HEp-2 cells were cultured in RPMI 1640 medium (Biochrome, Germany) supplemented with 10% fetal bovine serum (FBS, Biochrome, Germany) and 1% penicillin/streptomycin (P/S; 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin, Biochrome, Germany). The cells were maintained in an incubator at 37\u0026deg;C with 5% CO₂.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. KRAS Plasmid Constructs and Transfection to HEp-2 Cells\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAs described in our previous study [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], plasmid constructs carrying \u003cem\u003eKRAS\u003c/em\u003e-G12V mutation with and without LCS6 alteration were generated. Plasmid constructs were generated, containing a G12V mutant coding sequence (CDS) region and a normal LCS6 region of the \u003cem\u003eKRAS\u003c/em\u003e gene (\u003cem\u003eKRAS\u003c/em\u003e-CDSm-LCS6n) and a G12V mutant CDS region and a mutant LCS6 region of the \u003cem\u003eKRAS\u003c/em\u003e gene (\u003cem\u003eKRAS\u003c/em\u003e-CDSm-LCS6m) were constructed using the pLenti-CMV-GFP-2A-Puro (8950 bp) bi-cistronic lentiviral vector (Applied Biological Materials Inc., Canada). The constructs, including the empty plasmid, were transfected into HEp-2 cells using the amphotropic retroviral packaging cell line 2A (ATCC\u0026reg; CRL 12013, USA). For this purpose, 2A cells were transfected with a mixture of the constructs and Lipofectamine\u0026reg; 2000 (Invitrogen, USA). The constructs were subsequently packaged into viral particles and transferred to the cancer cells. Transfected HEp-2 cells were treated with 0.2 \u0026micro;g/ml puromycin to eliminate non-transfected cells, achieving a transfection efficiency of approximately 80\u0026ndash;90% after one month. To simplify the tracking of different HEp-2 cell types containing plasmid constructs in the experimental systems, the following abbreviations were assigned:\u003c/p\u003e\u003cp\u003eHEp2-0: Cells without plasmid transfection,\u003c/p\u003e\u003cp\u003eHEp2-1: Cells containing pLenti-\u003cem\u003eKRAS\u003c/em\u003e-CDSm-LCS6m plasmid construct,\u003c/p\u003e\u003cp\u003eHEp2-2: Cells containing pLenti-\u003cem\u003eKRAS\u003c/em\u003e-CDSm-LCS6n plasmid construct,\u003c/p\u003e\u003cp\u003eHEp2-3: Cells containing pLenti-CMV-GFP-2A empty plasmid vector.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Isolation of Cancer Stem Cell (CSC)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eStem cells were also isolated to generate xenograft mouse models expressing pLenti-\u003cem\u003eKRAS\u003c/em\u003e-CDSm-LCS6n and pLenti-\u003cem\u003eKRAS\u003c/em\u003e-CDSm-LCS6m. Control cells, along with cells containing plasmid constructs and empty plasmids, were trypsinized (Biochrome, Germany) and washed with 1x phosphate-buffered saline (PBS) (Biochrome, Germany) prior to antibody labeling. The cells were then incubated with CD44 (APC-conjugated) and CD24 (PE-conjugated) antibodies (Thermo Scientific, USA) in 100 \u0026micro;l of 2% fetal bovine serum (FBS) in Hank's Balanced Salt Solution (HBSS). Incubation was carried out for 1 hour at 4\u0026deg;C in the dark, with gentle agitation every 10 minutes. The cells were washed twice; first with 3 ml of 2% FBS/HBSS to remove unbound antibodies, and then with HBSS alone. The final cell pellet was resuspended in 400 \u0026micro;l of 2% FBS/HBSS. Cell sorting was performed by using a FACS Aria III device (BD Pharmingen, USA) to isolate the desired populations.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Preparation of Cells for Xenograft Model\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo create a xenograft model, both total cells and stem cells were transferred to mouse groups. HEp2-0, HEp2-1, HEp2-2, and HEp2-3 cells were detached from the plates using Trypsin/EDTA (0.25%, Biochrome, Germany) and washed with the complete medium. All cell groups, including isolated stem cell groups, were counted using trypan blue dye (Gibco, USA). 9.5x106 cells for total cells (TCs) and 3.2x105 cells for isolated stem cells (CSCs) were counted and suspended in 200 \u0026micro;l medium for each group. Then, 50 \u0026micro;l matrigel (STEMCELL Technologies, Canada) was added to each cell group on the ice.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Xenograft Mouse Experiments\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn the present study, 4-6-week-old CD1 nude male mice were used. The study was approved by Selcuk University Animal Experiments Local Ethics Committee (2015-00015). Mice were obtained from Kobay Experimental Animals Laboratory Inc. (Ankara, Turkiye) and Yıldırım Beyazıt University Experimental Application and Research Center (Ankara, Turkiye). The experiments were carried out in Individually Ventilated Cages (IVC) specifically designed for CD1 nude mice at Selcuk University Experimental Medicine Research and Application Center. All procedures were performed in accordance with humane and ethical treatment protocols to ensure comfort and minimal pain. All mice were housed at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50% humidity and a 12-hour light-dark cycle. Throughout the study, mice had \u003cem\u003ead libitum\u003c/em\u003e access to sterilized or disinfected tap-water and standard foot pellets. Bedding and litter trays were changed twice a week. The mice were kept under the aforementioned conditions for 2 weeks and the experiments began with animals weighing 18\u0026ndash;20 g.\u003c/p\u003e\u003cp\u003eThe animals were initially divided into two groups according to tumor injection sites: intraperitoneal (IP) and subcutaneous (SC) using a 26-gauge needle (Beybi, Turkiye) under sterile conditions. Each group was then further subdivided into four subgroups based on the transferred cell types (HEp2-0, HEp2-1, HEp2-2, HEp2-3). Additionally, one group was designated as a control without any procedure.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Tumor Monitoring\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn the groups treated with cells via the SC route, all tumor progression was monitored by inspection and palpation of the application site twice a week. In contrast, since palpation was not feasible, the groups receiving cells by the IP route were evaluated by observing the general health status and changes in body weight of the mice. The body weight of all mice and the tumor masses (width x length) in the SC-transplanted mice were systematically measured and recorded. These measurements were performed twice a week by the same investigator.\u003c/p\u003e\u003cp\u003eOn 40th day, determined by the general condition of the mice, the mice were sacrificed in the CO2 chamber. Tumor tissues and the other organs were collected for histopathological examination.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Histopathological Analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor histopathological examination, tumor tissues and other organs were fixed in 10% buffered formalin overnight at 4\u0026deg;C and embedded in paraffin. Paraffin blocks were sectioned at a thickness of 3\u0026ndash;4 microns using a microtome, and slides were prepared. After deparaffinization, slides were stained with Hematoxylin and Eosin (H\u0026amp;E) and examined under a bright-field microscope (Nikon, Japan).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Statistical Analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAll statistical analyses of the data obtained were performed with GraphPad Prism\u0026reg; V.10.4.1 software (GraphPad Software Inc., USA). Student's t-test and one-way ANOVA were used for group comparisons. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Transfection of plasmid constructs into the cells\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eAs demonstrated in our previous study [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e], two methods were utilized to confirm the successful transfection of plasmid constructs into the cells. First, the transfected cells were observed under a fluorescence microscope, as the plasmids contained a green fluorescent protein (GFP) marker. Second, the expression of non-mutant and mutant \u003cem\u003eKRAS\u003c/em\u003e (G12V-specific) proteins were evaluated via western blot analysis. The results revealed significantly higher levels of mutant KRAS protein in HEp2-1 and HEp2-2 cells compared to HEp2-0 and HEp2-3. These findings validate the successful transfection of G12V mutant \u003cem\u003eKRAS\u003c/em\u003e plasmid constructs into HEp2 cells.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Tumor Formation and Monitoring\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eFor the optimization study, the mice were divided into groups as described above, and either TCs or CSCs were transplanted into separate groups of mice. Both cell groups were administered via SC or IP routes to different groups of mice. While 9.5x10\u003csup\u003e6\u003c/sup\u003e cells were used for the TC injection, 3.2x10\u003csup\u003e5\u003c/sup\u003e cells were used for the CSC injection. In all cell-transplanted mice, tumor formation was observed; however, there was no significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in tumor size between TC- and CSC-transplanted mice. In terms of tumor development, the effect of approximately 3\u0026times;10⁵ HEp2 CSCs was found to be equivalent to the effect of 10\u0026times;10⁶ HEp2 TCs. Therefore, the subsequent experiments were performed using mice transplanted with TCs.\u003c/p\u003e\n \u003cp\u003eIn each group transplanted with cells, weight gain was observed until day 30\u0026ndash;33, followed by a significant weight loss from day 34\u0026ndash;38 (Fig. 1a). On the 3rd day after cell transplantation, masses were observed at the injection sites in all mice that received tumor cells via the SC route (Fig. 1b). However, because the masses had not yet reached a measurable palpable hardness, measurements of the masses were initiated on day 14. Our results showed that the G12V mutation increases the growth rate of the primary tumor, and the presence of the LCS6 alteration further accelerates tumor growth (Fig. 1c).\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eOn the 40th day, the weights of the primary tumor masses excised from the sacrificed mice were measured (Figs.\u0026nbsp;1d, 1e, 1f). Accordingly, when all cell types were evaluated together, the primary tumor masses grew faster in SC-transplanted mice compared to IP-transplanted mice.\u003c/p\u003e\n \u003cp\u003eThe mice were dissected, and the abdomen, thoracic cavity and skull were opened for macroscopic examination. No macroscopic metastasis was observed in any of the SC-transplanted mice. IP-transplanted mice, metastases were observed in mice injected with HEp2-1 and HEp2-2 cells, whereas no macroscopic metastases were detected in mice injected with HEp2-0 or HEp2-3 cells. In the group injected with HEp2-1 cells, tumor development showed a greater spread within the abdominal region compared to HEp2-2 (Figs.\u0026nbsp;1g, 1h).\u003c/p\u003e\n \u003cp\u003eThis section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Histopathological assessment\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eAfter dissection, the primary tumor tissues, potential metastatic sites, and suspected tissues were histopathologically examined. No metastasis was detected in mice transplanted with HEp2-0 and HEp2-3 cells, whereas metastases were observed in cells carrying \u003cem\u003eKRAS\u003c/em\u003e-G12V mutation. Among these, only liver metastasis (50% of cases) was detected in mice transfected with HEp2-2 cells carrying only the G12V mutation, whereas in mice transfected with HEp2-1 cells carrying also the G12V\u0026thinsp;+\u0026thinsp;LCS6 alteration, lung (75%), kidney (25%) and regional lymph node (75%) metastases were detected in addition to liver metastases. It was also observed that LCS6 alteration increased the frequency of liver metastases (100%). No brain metastases were observed in any cell group (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. 2a-e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMetastatic status in mice with tumor development.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHEp2 Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNumber of Mice with Metastases / Tumors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMouse No\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eOrgan/Tissue Metastasis Condition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePrimary Tumor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKidney\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBrain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRegional\u003c/p\u003e\n \u003cp\u003eLymph Node\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eHEp2-0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHEp2-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHEp2-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHEp2-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eGenetic alterations provide significant insights into the etiology and clinical progression of cancer. In particular, nucleotide changes in proto-oncogenes play an increasingly critical role in cancer diagnosis and treatment. One of the most important proto-oncogenes, the \u003cem\u003eKRAS\u003c/em\u003e gene, is notable not only for mutations within its coding region but also for alterations in its 3\u0026rsquo;UTR. These alterations possess substantial clinical relevance in the field of oncology [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn cancer research, the xenograft mouse model is a widely utilized method to examine the growth and metastatic behavior of cancer cells in a living biological system. In the literature, both TC and CSC applications are employed to develop xenograft mouse models. While the CSC xenograft model is used for studying the hematopoietic system and certain cancers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], the TC model is frequently utilized in laryngeal carcinoma research [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In our study, both cell types were used, and tumor development was observed in both models. This finding aligns with the stochastic model that posits that all tumor cells can contribute to cancer development. In the literature, the number of injected cells for TC applications ranges between 1\u0026ndash;10\u0026times;10⁶, while for CSC applications, 1\u0026ndash;5\u0026times;10⁵ cells are sufficient for cancer development [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These values are consistent with the cell counts used for tumor formation in our study.\u003c/p\u003e\u003cp\u003eGiven that the mass is not detectable by palpation in IP-transplanted mice, the SC transplantation method is frequently used in the literature for tumor size monitoring. Our finding that the IP method is more effective for metastasis tracking aligns with previous studies. In their xenograft mouse model study using a colorectal cancer cell line, Taibi, et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] observed no metastasis in mice that received SC injections, whereas metastases were detected in those that underwent IP injections. Similarly, in our study, no metastases were observed in mice that received SC injections, whereas both macroscopic and histopathological metastases were detected in mice that underwent IP injections. This finding is consistent with the existing literature.\u003c/p\u003e\u003cp\u003eIn the literature, the sacrifice timing in xenograft mouse model studies varies depending on the type of cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For aggressive cancers, this period can be as short as 14\u0026ndash;21 days [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], whereas in less aggressive, non-metastatic cancers or in mice receiving hematopoietic cell transplants, it can extend up to six months [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In our study, the optimal dissection time for HNSCC was determined to be day 40, based on the cessation of weight gain and the onset of weight loss in mice. In the literature, tumor formation at the injection site can be observed within the first week in xenograft mouse models of cancer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Similarly, in our study, the development of tumors was detected in all mice by day 3 following SC cell transplantation.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eKRAS\u003c/em\u003e-G12V mutation has been known to play a pivotal role in the development of cancer [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the present study, it was observed that this specific mutation led to a substantial augmentation in the primary tumor's growth rate.\u003c/p\u003e\u003cp\u003eThe other \u003cem\u003eKRAS\u003c/em\u003e alteration analyzed in our study, the T\u0026thinsp;\u0026gt;\u0026thinsp;G substitution in 3'UTR-LCS6 (rs61764370), has been linked to multiple cancers, including ovarian cancer [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], breast cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], lung cancer [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and HNCs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, the impact of this variation differs across various cancer types. Crowley, et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] investigated the rs61764370 variant in the SW48 colorectal cancer cell line and found that while it did not affect \u003cem\u003eKRAS\u003c/em\u003e expression, it reduced let-7 levels and increased cell proliferation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In contrast, Hollestelle, et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] reported no significant effect of this variant in ovarian and breast cancers [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In our study, we observed that while the G12V mutation alone promoted tumor growth in laryngeal cancer, the co-occurrence of the G12V mutation with the LCS6 variant further enhanced tumor growth (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eKRAS\u003c/em\u003e-LCS6 (rs61764370) variant is known to play a role in cancer progression; however, there are only a limited number of studies investigating its impact on metastasis. While previous observational studies have suggested that this variant increases metastatic potential in colorectal cancer, osteosarcoma, and gallbladder cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], no significant \u003cem\u003ein vivo\u003c/em\u003e studies have been conducted on this subject. In our study, we examined the in vivo effects of both the G12V and LCS6 mutations. Our findings indicate that in the presence of the G12V mutation alone, metastasis was observed only in the liver (50% of cases). However, when both the G12V and LCS6 mutations were present, metastases were detected in the liver (100%), lungs (75%), kidneys (25%), and regional lymph nodes (75%). Notably, no brain metastases were observed in any group. Given that HNSCC predominantly spreads locally, brain metastases are rare [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This characteristic likely explains the absence of brain metastases in any of the mice in our present study. Ulusan, et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] reported that the rs61764370 variant is associated with an increased risk of lymph node metastasis in HNCs. Similarly, in our study, the group carrying the LCS6 variant exhibited a high incidence of lymph node metastasis, providing \u003cem\u003ein vivo\u003c/em\u003e validation for the clinical observation. Furthermore, in our previous \u003cem\u003ein vitro\u003c/em\u003e cell model study reported that the LCS6 variant had no additional effect on tumor growth or invasion capacity beyond the G12V mutation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, our \u003cem\u003ein vivo\u003c/em\u003e mouse model study demonstrated a significant difference, highlighting the importance of complementing \u003cem\u003ein vitro\u003c/em\u003e studies with \u003cem\u003ein vivo\u003c/em\u003e research in cancer investigations.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study demonstrates that, in xenograft mouse model, the \u003cem\u003eKRAS\u003c/em\u003e-G12V mutation increases HNSCC tumor growth and metastasis at relatively low rates, whereas in the combination with the LCS6 alteration, both tumor growth and metastasis are significantly increased. The LCS6 alteration increases specifically metastasis to the liver, lungs, kidneys, and regional lymph nodes. In conclusion, our findings make a significant contribution to the literature on the impact of \u003cem\u003eKRAS\u003c/em\u003e-G12V and LCS6 alterations on cancer progression and metastasis. They may also serve as a guide for future studies aimed at developing new therapeutic strategies targeting these alterations. Furthermore, the difference between our \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e results highlights the critical importance of \u003cem\u003ein vivo\u003c/em\u003e models in cancer research to better understand the progression of tumors and clinical outcomes.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used in this manuscript:\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHNCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHead and neck cancers\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHNSCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHead and neck squamous cell carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCancer stem cell\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTotal cell\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e3’UTR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3’ untranslated region\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLCS6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eLet-7 complementary site 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCoding sequence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSubcutaneous\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGFP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGreen fluorescent protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, H.A.; methodology, H.A., H.T. and K.O.; validation, H.A., M.B.K., V.B.Y. and Y.S.A.; formal analysis, H.A., M.B.K. and Y.S.A.; investigation, M.B.K., V.B.Y., Y.S.A., H.T. and K.O.; resources, H.A., M.B.K., H.T. and K.O.; data curation, H.A.; writing\u0026mdash;original draft preparation, M.B.K.; writing\u0026mdash;review and editing, H.A. and V.B.Y.; visualization, M.B.K., H.T. and K.O.; supervision, H.A.; project administration, H.A.; funding acquisition, H.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was partially funded by the Scientific and Technological Research Council of T\u0026uuml;rkiye (T\u0026Uuml;BİTAK), grant number 112S498.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Selcuk University Animal Experiments Local Ethics Committee (2015-00015 and 10.01.2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data supporting this study\u0026apos;s findings are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Professor Thomas E. Carey, University of Michigan, USA, for supplying the cell line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDisclaimer/Publisher’s Note:\u003c/strong\u003e The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. 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The let-7 microRNA binding site variant in KRAS as a predictive biomarker for head and neck cancer patients with lymph node metastasis. \u003cem\u003ePathology-Research and Practice \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e239\u003c/em\u003e, 154147.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"KRAS, G12V, rs61764370, xenograft mouse model, metastasis, HNSCC ","lastPublishedDoi":"10.21203/rs.3.rs-7398588/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7398588/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHead and neck cancers (HNCs) are the seventh most common cancers worldwide, with \u003cem\u003eKRAS\u003c/em\u003e mutations playing a key role for developing and progression of cancer. The G12V mutation is one of the most commonly identified KRAS mutation and has been associated with tumor progression and aggressive disease. Additionally, T\u0026thinsp;\u0026gt;\u0026thinsp;G alteration in the binding site of miRNA let-7 of the \u003cem\u003eKRAS\u003c/em\u003e-3'UTR (rs61764370), let-7 complementary site 6 (LCS6), has been found to promote cancer development, metastasis, and treatment resistance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMaterial and Methods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHEp2 laryngeal cancer cells, transfected with plasmid constructs carrying the \u003cem\u003eKRAS\u003c/em\u003e-G12V mutation and the combined G12V\u0026thinsp;+\u0026thinsp;LCS6 mutations were transplanted subcutaneously (SC) or intraperitoneally (IP) into CD-1 nude mice with non-transfected control cells. The mice were euthanized on the 40th day post-transplantation and examined histopathologically.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrimary tumor development was observed in all cell-transplanted mice. The G12V mutation significantly promoted tumor growth (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and LCS6 alteration further accelerated progression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No metastases occurred in SC-injected mice, but IP-injected mice with \u003cem\u003eKRAS\u003c/em\u003e mutations developed metastases. G12V alone led to only liver metastases (50%), whereas G12V\u0026thinsp;+\u0026thinsp;LCS6 resulted in metastases in the liver (100%), lung (75%), kidney (25%), and paraaortic lymph nodes (75%). No brain metastases were observed in any group.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study shows that while the \u003cem\u003eKRAS\u003c/em\u003e-G12V mutation enhances HNSCC tumor growth and metastasis at relatively low rates, its combination with the LCS6 alteration significantly accelerates both. These findings suggest that miRNAs and miRNAs binding site mutations in \u003cem\u003eKRAS\u003c/em\u003e-3\u0026rsquo;UTR contribute to cancer aggressiveness and could serve as potential therapeutic targets.\u003c/p\u003e","manuscriptTitle":"Investigation of Combined Effects of KRAS-G12V and KRAS- 2 LCS6 (rs61764370) Alterations on Tumor Progression and Me- 3 tastasis in HNSCC by Using Xenograft Mouse Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-26 02:07:09","doi":"10.21203/rs.3.rs-7398588/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e27646b-a8bc-45d1-8df5-6858bfacf07e","owner":[],"postedDate":"October 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-04T04:23:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-26 02:07:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7398588","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7398588","identity":"rs-7398588","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}