High-fat Diet Enhances the Growth of Renal Cell Carcinoma and Alters Immune Cells in Spleen, Kidney and Tumor

doi:10.21203/rs.3.rs-3149902/v1

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High-fat Diet Enhances the Growth of Renal Cell Carcinoma and Alters Immune Cells in Spleen, Kidney and Tumor

https://doi.org/10.21203/rs.3.rs-3149902/v1

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Renal cell carcinoma (RCC) is strongly associated with abnormal or excessive fat deposition in the body, whose processes include persistent adipose inflammation and other disturbances with the development and function of immune cells. Researchers have recently become more and more interested in understanding how high-fat diet (HFD) affects the development and course of RCC by causing immunological dysfunction. The changes of immune cell groups in RCC, particularly those in normal kidneys and tumors, are, nevertheless, still poorly understood. Consequently, we explore the effect of HFD on the changes of immune cell groups in spleens, normal kidneys and tumors, mainly revealing the changes of T cells, B cells and NK cells, and further preliminarily exploring the changes of NK cell phenotype. Our findings demonstrate that: 1) HFD speeds up the growth of ACHN tumors; 2) HFD increases the frequency of CD45⁺ live cell, T cell and cNK in spleen, increases the frequency of T cell, NK cell and cNK in normal kidney, as well as increases the frequency of CD45⁺ live cell, NK cell and cNK in tumor;3) HFD decreases the frequency of B cell, NK cell and ILC1 in spleen, decreases the frequency of CD45⁺ live cell, B cell, and ILC1 in normal kidney, as well as decreases the frequency of T cell, B cell and ILC1 in tumor. These data will open up new avenues for immunotherapy in individuals with obese renal cell carcinoma.

T cell

B cell

NK cell

NK cell phenotype

high-fat diet

renal cell carcinoma

BALB/c-nude

Obesity and overweight are both described as abnormal or excessive fat deposition that could be harmful to one's health. The body mass index (BMI) is calculated by dividing a person's height in m² by their weight in kg 1^,2. According to the World Health Organization, those with a BMI of at least 25 kg/m² are considered overweight, while those with a BMI of at least 30 kg/m² are considered obese 2. Almost 1.9 billion persons aged 18 and older were expected to be overweight in 2016, with over 650 million of those adults being obese. From 1975 and 2016, the prevalence of obesity nearly tripled globally 3^,4. Inappropriate or excessive fat buildup in body is intimately associated to the elevated risk of many cancer kinds 5, like endometrial cancer 6^,7, postmenopausal breast cancer 8-11, colorectal cancer 8^,12-14, esophageal cancer 15, renal cancer 16^,17, meningioma cancer, pancreatic cancer 18^,19, gastric cardia cancer, liver cancer 20^,21, ovarian cancer 22, gallbladder cancer 23, and thyroid cancer 24-26. Several factors are involved in the tumor promoting effect of obesity, including persistent adipose inflammation and other disturbances with the development and function of immune cells 27. When the prevalence of overweight and/or obesity increases globally, more and more researchers are studying the mechanisms that induce immune dysfunction and affect cancer progression and treatment.

Renal cell carcinoma (RCC) accounts for 5% and 3% of all tumor diagnosis globally, making it the sixth most prevalent cancer in men and the tenth most common in women 28. Obesity/overweight, hypertension, gender, cigarettes, and chronic kidney disease are risk factors for RCC 29. Recent research has demonstrated that the tumor microenvironment is crucial to the pathogenesis, treatment, and carcinogenesis of RCC 30. The most prevalent RCC histologic subtype, clear cell RCC (ccRCC), typically has distinctive second-hit function deletion mutations in von Hippel-Lindau (VHL) against the backdrop of deletion of chromosome 3p, where VHL is located 30^,31. Moreover, ccRCC have a significant level of immune cell infiltration, especially from T cells 30^,32.

Peripheral blood vessels, immune cells, fibroblasts, inflammatory cells generated from bone marrow, different signal molecules, and extracellular matrix (ECM) make up the milieu known as the tumor microenvironment (TME), which surrounds tumor cells 33. TME is a challenging setting for tumor cells to thrive and proliferate, and immune cells therein and how they regulate themselves have a significant impact on the formation and progression of malignancies 33-35. TME is one of physiological conditions that affects T cell metabolism. Cancer cells' limited nutrition uptake and accumulation of waste products can directly alter T cell physiology through the production of inhibitory ligands, which in turn severely restricts T cell metabolism. Effective immunotherapies may be hindered by this regulation of T cell metabolism, which may reduce the activity of effector T cells and increase the number of suppressive regulatory T cells 36. Depending on the type of antigens they identify and the make-up of the TME, B lymphocytes can play a variety of intricate roles in tumors. B cells can deliver antigens to T cells from immune complexes that are endocytosed by dendritic cells in tumors with mature tertiary lymphoid structures (TLS) rich in germinal centers. This is especially useful in tumors with weak immunogenicity that are unable to directly activate T cells. Plasma cell (PC) produced antibodies may also encourage macrophage and natural killer (NK) cell anti-tumor effector capabilities. In contrast, B cells take on a regulatory nature and suppress immune responses in tumors with immature TLS missing a germinal core 37. One of the main limitations on NK cell activation may be the immunosuppressive TME. The metabolism of NK cells is disturbed in the TME, which may restrict the effector capabilities of NK cells due to the lack of nutrients and oxygen and the greater concentration of tumor-derived metabolic end products such as lactate . It is understood that tumor cell metabolism affects the regional metabolic environment by suppressing anti-tumor immunity 38. The precise process by which systemic metabolism affects local metabolism inside the TME is still not fully known 39. It is also not described the effect of changes in systemic metabolism generated by obesity affect immune cells in the local TME.

Here, taking RCC as an example, we investigate how high-fat diet (HFD) affects the changes of immune cells in TME. We employ the heterologous mouse tumor model to investigate the changes of immune cells brought on by HFD in spleens, normal kidneys, and tumors with flow cytometry, primarily revealing the changes in T cells, B cells and NK cells, and then initially exploring the changes in NK cell phenotype. The findings demonstrate that: 1) HFD speeds up the growth of ACHN tumors; 2) HFD increases the frequency of CD45⁺ lymphocyte, T cell and cNK (CD49b⁺) in spleen, increases the frequency of T cell, NK cell and cNK (CD49b⁺) in normal kidney, as well as increases the frequency of CD45⁺ lymphocyte, NK cell and cNK (CD49b⁺) in tumor;3) HFD decreases the frequency of B cell, NK cell and ILC1 (CD49a^-) in spleen, decreases the frequency of CD45⁺ lymphocyte, B cell, and ILC1 (CD49a^-) in normal kidney, as well as decreases the frequency of T cell, B cell and ILC1 (CD49a^-) in tumor. These data will open up new avenues for immunotherapy in individuals with obese renal cell carcinoma.

2.1 Cell lines and Culture

Procell Biosciences (Wuhan, China) supplied the ACHN cells, which were kept alive in RPMI 1640 Medium with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin. Every cell was cultivated at 37°C in an incubator with 5% CO₂.

2.2 Experimental Animal Model

Three-week-old male BALB/c mice were bought from Guangzhou Xinyi Biotechnology Co., Ltd. Three-week-old mice were used in all trials, and they were assigned into either the normal fat diet group (NFD) with 10% kcal fat; n = 4) or the high fat diet group (HFD with 60% kcal fat; n = 4). They were fed NFD or HFD for 20 weeks. The Guangdong Academy of Agricultural Sciences' Animal Health department operated a single animal facility where all mouse colonies and experimental animals were housed in specific pathogen-free conditions. All animals were employed in conformity with the Guangdong Academy of Agriculture Sciences' Animal Health department's standards for animal care. The Guangdong Academy of Agriculture Sciences' Animal Health division gave its approval to each mouse experiment.

2.3 Mouse RCC Tumor Models

Mice were subcutaneously injected with 2X10⁶ ACHN cells in the abdomen flank after 20 weeks of NFD or HFD diet. Tumors were measured using a caliper every three days until they became palpable. 0.5 x D x d², where D is the long diameter and d is the short diameter, is the formula used to compute tumor volumes. After 18 days, the mice were anesthetized, removed and weighed their tumors. The tumor, normal kidney, and spleen tissues were then either sliced and preserved in formalin or liquid nitrogen or processed into a single-cell suspension for further examination.

2.4 Flow cytometry analysis

Using ophthalmic scissors, the spleen was divided into pieces and then resuspended in PBS to prepare the sample. RBC Lysis Buffer (BL503A, Biosharp) was used to lyse red blood cells after a 10-minute incubation in the dark at room temperature. After cutting the tumor and normal kidney with ophthalmic shears, the tissues were digested for 30 minutes at 37 °C in RPMI 1640 medium with 2% FBS, 2 mg/mL collagenase IV (BioFroxx, 1904GR001), and 2 mg/mL DNase I (BioFroxx, 1121MG010). After digestion, the cell suspensions were filtered using a filter with a 70-m mesh size. The cells were then made into single-cell suspensions after being washed twice with PBS.

Single-cell suspensions obtained from spleen, normal kidney, or tumor were stained with surface marker antibodies for cell analysis on an Attune NxT flow cytometer (ThermoFisher, 1800200S). The relative antibodies were shown as followed. Becton Dickinson provided the FITC-conjugated anti-mouse CD45 (553079), APC-Cy7-conjugated anti-mouse CD3 (560590), and BV510-conjugated mouse CD19 (562956) antibodies. We bought the following antibodies from Biolegend: AF647-conjugated anti-mouse NKp46 (137628), PE-Cy7-conjugated anti-mouse CD49a (142607), and PE-conjugated anti-mouse CD49b (DX5, 108907). Becton Dickinson sold us 7-Aminoactinomycin D (559925) and Live/Dead Green dye (553141). Software called FlowJo 10 was used for data analysis. Figure 2 illustrates the method used to gate CD45⁺living cells, CD3⁺T cells, CD19⁺B cells, NKp46⁺NK cells, ILC1, and cNK.

2.5 Statistical Analysis

The GraphPad Prism software, version 8.0, was used to perform all charting. The outcomes are shown as mean standard deviation (M SD). To compare variables normally distributed, the independent-sample T test was performed. When the P value was less than 0.05, differences were deemed statistically significant. Using the SPSS program, the statistical analysis was completed (release 13.0).

3.1 HFD accelerates ACHN tumor growth

We randomly assigned three-week-old BALB/c mice to either the NFD group (NFD, containing 10% kcal of fat; n=4) or the HFD group (HFD, containing 60% kcal of fat, n=4) and fed them with NFD or HFD for 20 weeks (Figure 3A). We find that the weight of the mice fed the HFD increases significantly compared to the mice fed the NFD after 20 weeks (P<0.05) (Figure 3B).

To develop a xenogeneic animal tumor model, mice were subcutaneously injected with ACHN cells after 20 weeks of feeding. We discover that the tumor volume in mice treated with HFD develop more quickly than that in mice treated with NFD after 18 days of ACHN cell injection (P 0.05) (Figure 3C). After 18 days, we killed the mice and obtained their tumor tissue for weighing. We find that the tumor weight of mice fed HFD is considerably higher than that of mice fed NFD (P<0.05) (Figure 3D).

3.2 CD45⁺ live cell change in Spleen, Kidney, and Tumor

As shown in figure 4, HFD-treated mice have a higher frequency of CD45⁺ live cell than NFD-treated mice in spleen and tumor, while HFD-treated mice have a lower frequency of CD45⁺ live cell than NFD-treated mice in normal kidney. In spleen of HFD-treated mice group, ~90.6% of immune cells are CD45⁺ live cells, which rises by nearly one-fold than that in NFD-treated mice group (~40.2%) (Figure 4A-4B). In tumor of HFD-treated mice group, ~30.0% of immune cells are CD45⁺ live cells, which gives rise to 29 times as many nearly than that in NFD-treated mice group (~1.03%) (Figure 4E-4F). In contrast, in normal kidney of HFD-treated mice group, ~1.08% of immune cells are CD45⁺ live cells, which is lower than that in NFD-treated mice group (~6.40%) (Figure 4C-4D). Figure 4G-I expresses the trend of CD45⁺live cells in the spleen, normal kidney, and tumor with greater consistency.

3.3 CD3⁺T cells and CD19⁺B cells change in Spleen, Kidney, and Tumor

As illustrated in figure 5, HFD-treated mice have a higher frequency of CD3⁺T cells than NFD-treated mice in spleen and normal kidney, while HFD-treated mice have a lower frequency of CD3⁺T cells than NFD-treated mice in tumor. In spleen of HFD-treated mice group, ~6.59% of CD45⁺live cells are CD3⁺T cells, which rises by almost twelve-fold than that in NFD-treated mice group (~0.53%) (Figure 5A-5B). In normal kidney of HFD-treated mice group, ~5.84% of CD45⁺live cells are CD3⁺T cells, which nearly gives rise to 3 times than that in NFD-treated mice group (~1.95%) (Figure 5C-5D). In contrast, in tumor of HFD-treated mice group, ~27.0% of CD45⁺live cells are CD3⁺T cells which is lower than that in NFD-treated mice group (~54.4%) (Figure 5E-5F).

As displayed in figure 5, HFD-treated mice have a lower frequency of CD19⁺B cells than NFD-treated mice in spleen, normal kidney and tumor. In spleen of HFD-treated mice group, ~18.1% of CD45⁺live cells are CD19⁺B cells, which is significantly lower than that in NFD-treated mice group (~24.4%) (Figure 5A-5B). In normal kidney of HFD-treated mice group, ~2.96% of CD45⁺live cells are CD19⁺B cells, which is nearly 2 times lower than that in NFD-treated mice group (~6.98%) (Figure 5C-5D). In tumor of HFD-treated mice group, ~0.30% of CD45⁺live cells are CD19⁺B cells which is nearly 10 times lower than that in NFD-treated mice group (~3.23%) (Figure 5E-5F). Moreover, figure 5G-I significantly expresses the trend of CD3⁺T cells and CD19⁺B cells in the spleen, normal kidney, and tumor.

3.4 NKp46⁺NK cells change in Spleen, Kidney, and Tumor

NK cells express both NK1.1 and NKp46 cell markers in C57BL/6 mice, whereas NK cells only express NKp46 in BALB/C mice 40. Therefore, in this experiment, we use NKp46 as a cell marker for NK cells. As depicted in figure 6, HFD-treated mice have a higher frequency of NKp46⁺NK cells than NFD-treated mice in normal kidney and tumor, while HFD-treated mice have a lower frequency of NKp46⁺NK cells than NFD-treated mice in spleen. In normal kidney of HFD-treated mice group, ~16.8% of CD45⁺live cells are NKp46⁺NK cells, which nearly gives rise to 2.7 times than that in NFD-treated mice group (~6.23%) (Figure 6C-6D). In tumor of HFD-treated mice group, ~7.92% of CD45⁺live cells are NKp46⁺NK cells which rises by almost two-fold than that in NFD-treated mice group (~3.69%) (Figure 6E-6F). In contrast, in spleen of HFD-treated mice group, ~5.17% of CD45⁺live cells are NKp46⁺NK cells, which is nearly 9.8 times lower than that in NFD-treated mice group (~50.7%) (Figure 6A-6B). Additionally, figure 6G-I significantly expresses the trend of NKp46+NK cells in the spleen, normal kidney, and tumor.

3.5 NK cell phenotype change in Spleen, Kidney, and Tumor

According to studies, NK cells that reside in tissue primarily express CD49a while NK cells that circulate in the blood primarily express CD49b 41-45. In this study, we characterized the NK cell phenotype of CD49a^- as type 1 innate immune cell (ILC1) and CD49b⁺ as circulating NK cell (cNK). We discover that the diet has a significant impact on the frequency of cNK and ILC1NK cell subsets in this experiment. As can be seen in figure 7, HFD-treated mice have a higher frequency of cNK cells than NFD-treated mice in spleen, normal kidney and tumor, while HFD-treated mice have a lower frequency of ILC1 cells than NFD-treated mice in spleen, and normal kidney and tumor. In spleen of HFD-treated mice group, ~46.4% of NKp46⁺NK cells are cNK cells, which nearly gives rise to 10 times than that in NFD-treated mice group (~4.39%) (Figure 7A-7B); in normal kidney of HFD-treated mice group, ~43.9% of NKp46⁺NK cells are cNK cells, which nearly gives rise to 1.5 times than that in NFD-treated mice group (~29.2%) (Figure 7C-7D); and in tumor of HFD-treated mice group, ~74.5% of NKp46⁺NK cells are cNK cells, which nearly gives rise to 3 times than that in NFD-treated mice group (~25%) (Figure 7E-7F). On the contrary, in spleen of HFD-treated mice group, ~48.4% of NKp46⁺NK cells are ILC1 cells, which nearly decreases by 1.9 times than that in NFD-treated mice group (~94.9%) (Figure 7A-7B); in normal kidney of HFD-treated mice group, ~47.9% of NKp46⁺NK cells are ILC1 cells, which nearly decreases by 1.3 times than that in NFD-treated mice group (~64.1%) (Figure 7C-7D); and in tumor of HFD-treated mice group, ~19.7% of NKp46⁺NK cells are ILC1 cells, which nearly decreases by 3.4 times than that in NFD-treated mice group (~66.7%) (Figure 7E-7F). In figure7G-I, we can intuitively see the trend of NK subsets in BALB/c-nude mice across multiple organs based on ILC1 and cNK expression in mice treated with NFD or HFD.

Cytokine therapy in RCC has gradually come under the spotlight in recent studies 46. It was consistently reported that 5–10% of patients who received this treatment experienced durable tumor regression 47^,48. In this study, we preliminary examine the change tendency in immune cells (including T cells, B cells and NK cells) of heterologous mouse RCC tumor model treated with HFD, which may provide better immune therapies for RCC patients with overweight and obesity.

4.1 T cells

Obesity with systemic metabolism changes plays a significant role in the TME and has an impact on anti-tumor immunity 49^,50. HFD-induced obesity impairs CD8⁺T cell function in the murine TME, accelerating tumor growth, as demonstrated by Alison E. Ringel et al.39. Under the influence of HFD, tumor cells were able to increase their fat uptake, but CD8⁺T cells that had already invaded the tumor were unable to do so. This resulted in altered fatty acid partitioning in HFD tumors and impaired CD8⁺T cell infiltration and function. Analysis of tumor-invading T cells from three treatment-naive ccRCC patients showed distinct expression changes in comparison to peripheral blood and normal renal parenchyma, according to Nicholas Borcherding et al. 51. According to our study, the analysis of tumor-invading T cells shows minimal expression changes when compared to peripheral blood and normal renal parenchyma from heterologous mouse RCC tumor model treated with HFD. Despite lacking T cells and having a thymus defect, BALB/c nude mice can still activate T cell expression when stimulated by human ACHN renal cancer cells. Our findings might imply that HFD can cause T cells to express differently, which would require additional research to prove.

4.2 B cells

Regulatory B cells (Breg) are known to be essential for preserving immune homeostasis and preventing the onset of obesity-related insulin resistance 52^,53. The CD19⁺CD1d⁺CD5⁺B cell subset is a distinct subset of powerful Breg cells with the ability to produce IL-10 and inhibit T cell responses, preserving homeostasis in adipose tissue 53-55. In obese patients, impaired Breg cell function will accelerate the development of adipose tissue inflammation. According to Beth A. Helmink's research on the function of B cells in RCC, memory B cells may serve as antigen-presenting cells that promote the development of both memory and naive tumor-associated T cell responses 56. Moreover, B cells have the ability to release a variety of cytokines, including TNF, IL-2, IL-6, and IFN-γ, which they use to recruit and activate other immune effector cells, such as T cells. In our investigation, we discover reduced frequencies of CD19⁺B cells that expressed themselves in spleens, normal kidneys, and tumors that had been given HFD. Our findings may imply that HFD can cause tumors by potentially affecting Breg cell function, although further research is required to confirm this.

4.3 NK cells and their phenotypes

NK cells can encourage the eradication of tumor and virus-infected cells. NK cells play a significant role in triggering tumorous cell death through the release of cytokines and chemokines as a component of the first line of defense against neoplastic development 57-60. Recent research has shown that NK cells, which make up 20% of immune cells and have a non-classical character, are present in RCC and that their frequency is directly connected with the prognosis of RCC . According to research by Julia Spielmann et al. 61, mice given the HFD have considerably lower frequencies of both the mature CD11b⁺CD27⁺NK cell subset and total NK cells than mice fed the NFD. Moreover, feeding HFD caused notable modifications in the expression of the NK cell maturation markers KLRG1 and CD127 61. In contrast to Julia Spielmann's research, we discover that mice fed the HFD had higher frequencies of NKp46⁺NK cells in normal kidneys and tumors and lower frequencies of NKp46⁺NK cells in spleens when compared to mice fed the NFD. In general, tumor-invading NK cells are sensitive to the inhibition of inhibitory receptor (IR) ligands and have the innate ability to detect altered cells under the activation of cytokines 62. After interacting with IRs, HFD may encourage the activation of NK cells that have infiltrated tumors and reduce their ability to kill, resulting in an impact that may increase tumor growth but still requires more experimental validation. According to the NK cell phenotypes, we discover that the frequency of the CD49b⁺CD49a^-NK cell subset was significantly higher in mice treated with HFD compared to mice treated with NFD, while the frequency of the CD49b^-CD49a^-NK cell subset was obviously lower in mice treated with HFD compared to mice treated with NFD.

However,

In conclusion, our results demonstrate that: 1) HFD can foster RCC development; 2) the frequency of tumor-infiltrating T cells treated with HFD was lower than treated with NFD, which may indicate that HFD can achieve the tumor-promoting effect by affecting the expression of T cells, which still needs further study to prove; 3) the frequency of spleen/tumor-invading CD19⁺B cells treated with HFD was lower than treated with NFD, which may indicate that HFD can achieve the tumor-promoting effect by affecting the expression of B cells. Thus, the HFD and immune cell alterations can be a potential therapy that could develop into another immunologic therapy for RCC brought on by obesity. It is necessary to do additional research to validate our findings and clarify the underlying molecular pathways by which HFD alters immune cells. However, our experimental findings only initially demonstrate the trend of immune cell population changes brought on by HFD. The statistical significance of changes in immune cell populations brought on by HFD will be investigated in the following experiment, along with additional immune cells, such as T cell subsets and macrophage populations.

Data Availability Statement

This article has included all original contributions of the study. Further questions can be addressed to the corresponding author.

Ethics Statement

The Guangdong Academy of Agricultural Sciences' Animal Health department examined and approved the animal study.

Conflict of interest

The authors affirm that there were no financial or commercial ties that might be viewed as having a potential conflict of interest in this research.

Author Contributions

With the assistance of MY, ZZR prepared and carried out experimental investigation. Then ZZR and MK executed the animal tests under supervision of MY. ZZR established the flow cytometric and workflow as well as analyzed the flow cytometry. The manuscript was primarily written by ZZR and MK with support from MY. MY revised the manuscript critically. The final manuscript was read and approved by all writers.

Acknowledgments

No.

Funding

This research was funded by National Natural Science Foundation of China (82270756); Natural Science Foundation of Guangdong, China (2018A030313527); The Basic and Applied basic research Foundation of Guangdong Province, China (2019A1515010176); The Science and Technology Project of Guangzhou, China (202102010133); The Science and Technology Project of Shenzhen, China (JCYJ20190808095615389).

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No competing interests reported.

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High-fat Diet Enhances the Growth of Renal Cell Carcinoma and Alters Immune Cells in Spleen, Kidney and Tumor

Status:

Version 1

Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Cell lines and Culture

2.2 Experimental Animal Model

2.3 Mouse RCC Tumor Models

2.4 Flow cytometry analysis

2.5 Statistical Analysis

3 Results

3.1 HFD accelerates ACHN tumor growth

3.2 CD45⁺ live cell change in Spleen, Kidney, and Tumor

3.3 CD3⁺T cells and CD19⁺B cells change in Spleen, Kidney, and Tumor

3.4 NKp46⁺NK cells change in Spleen, Kidney, and Tumor

3.5 NK cell phenotype change in Spleen, Kidney, and Tumor

4 Discussion

4.1 T cells

4.2 B cells

4.3 NK cells and their phenotypes

5 Summary

Declarations

References

Additional Declarations

Status:

Version 1

High-fat Diet Enhances the Growth of Renal Cell Carcinoma and Alters Immune Cells in Spleen, Kidney and Tumor

Status:

Version 1

Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Cell lines and Culture

2.2 Experimental Animal Model

2.3 Mouse RCC Tumor Models

2.4 Flow cytometry analysis

2.5 Statistical Analysis

3 Results

3.1 HFD accelerates ACHN tumor growth

3.2 CD45+ live cell change in Spleen, Kidney, and Tumor

3.3 CD3+T cells and CD19+B cells change in Spleen, Kidney, and Tumor

3.4 NKp46+NK cells change in Spleen, Kidney, and Tumor

3.5 NK cell phenotype change in Spleen, Kidney, and Tumor

4 Discussion

4.1 T cells

4.2 B cells

4.3 NK cells and their phenotypes

5 Summary

Declarations

References

Additional Declarations

Status:

Version 1

3.2 CD45⁺ live cell change in Spleen, Kidney, and Tumor

3.3 CD3⁺T cells and CD19⁺B cells change in Spleen, Kidney, and Tumor

3.4 NKp46⁺NK cells change in Spleen, Kidney, and Tumor