Lung Cancer Cells Secrete Glutamine to Accumulate Tumor-Associated Macrophages.

Introduction
Tumor-associated macrophages (TAMs), a significant component of the tumor microenvironment, are key contributors to cancer progression. They facilitate tumor growth and stress resistance through various mechanisms, including the secretion of IL-6 and TNF-α to enhance drug resistance,1 VEGFA to promote angiogenesis, and IL-10 to suppress anti-tumor immune responses.2,3 In non-small cell lung cancer (NSCLC), increased TAM infiltration has been observed in drug-resistant tissues,4,5 and TAMs further contribute to metastasis by promoting cancer cell migration.6,7
In healthy tissues, macrophages are highly plastic and responsive to environmental cues that regulate their infiltration, phenotype, and function.8,9 During wound healing, for example, inflammatory signals first recruit macrophages and monocytes, which clear debris and produce reactive oxygen species (ROS) to combat infection.8,9 As healing progresses, macrophages transition to pro-angiogenic phenotypes that secrete VEGF and later adopt immunosuppressive roles that promote tissue remodeling through the activation of regulatory T cells, secretion of growth factors and cytokines, and stimulation of collagen synthesis.8,9
Cancer cells exploit this macrophage plasticity to generate a tumor-promoting microenvironment by recruiting and polarizing TAMs toward pro-tumor phenotypes. This is achieved through context- and cancer type-specific secretion of cytokines, growth factors, metabolites, and nucleotides.10–13 Stress-tolerant and metastatic cancer cells, in particular, are adept at leveraging both intrinsic and extrinsic mechanisms to exploit TAMs and drive tumor progression.14 Given the critical role of TAMs in cancer development, understanding the molecular interactions between cancer cells and macrophages is essential for developing therapeutic strategies to disrupt these signaling networks. However, the mechanisms by which NSCLC cells promote TAM infiltration within the tumor microenvironment remain incompletely understood.
Our previous studies have shown that cancer cell expression of integrin αvβ3, a marker and driver of cancer stem-like properties, correlates with increased pro-tumor TAM infiltration across various cancer types, including NSCLC.15 Integrin αvβ3 is a key driver of NSCLC progression, promoting tumor initiation, drug resistance, and tolerance to metabolic and oxidative stress.16–18 However, its functional role in modulating the tumor microenvironment, particularly TAM recruitment, remains unclear. In this study, we tested the hypothesis that NSCLC cells use integrin αvβ3 to promote TAM accumulation within the tumor microenvironment, thereby enhancing tumorigenesis.

Materials and Methods

Study design
For in vivo and in vitro assays, sample sizes were calculated using power analysis, with effect size and standard deviation determined based on previously published similar assays15,17,19 and a power of 0.8 for each hypothesis using G*Power software. To avoid bias, numbers not associated with experimental information were assigned to all animals and specimens.

Cell lines
Mouse Louis Lung Carcinoma (LLC, CRL-1642, grown in DMEM) and human lung adenocarcinoma HCC827 (CRL-2868, grown in RPMI) cell lines were obtained from the American Type Culture Collection (ATCC). Upon receipt, each cell line was expanded and cryopreserved as low-passage stocks. All the cell lines were used within 40 passages. The cells were tested for mycoplasma using the MycoScope PCR Mycoplasma Detection Kit (Genlantis, MY01050) every other month. For ectopic expression and genetic knockdown, the cells were transfected with a vector control, integrin β3, or luciferase using a lentiviral system.

Flow cytometry
The cell pellets were washed with PBS, blocked with 1% BSA in PBS for 30 minutes at room temperature, incubated with or without anti-integrin αvβ3 antibody (Promega, MAB1976) for one hour at 4°C, stained with goat anti-mouse IgG antibody (Thermo, A21235) for 30 minutes at 4°C and incubated with propidium iodide (Sigma, P4864) for 5 minutes at room temperature. Flow cytometry was performed on a BD Fortessa X-20 (BD Biosciences) analyzer, and the data were analyzed using FlowJo (Treestar) software.

Immunohistochemistry
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded slides using ImmPRESS Excel Staining Kit (anti-rabbit) (Vector, MP-7601) or ImmPRESS HRP anti-rat IgG, mouse absorbed (peroxidase) polymer detection kit (Vector, MP-7444) following the manufacturer’s instructions. For both integrin β3 and F4/80, low-pH antigen and high-pH antigen retrieval were performed, respectively, for 20 minutes at 95°C. The slides were imaged using an Olympus VS200 Slide Scanner (Olympus). %F4/80-positive cell was measured using QuPath.20

Conditioned media collection
Cells were preincubated in RPMI with 5% charcoal-stripped FBS and 100 μM glutamine in suspension at 1 million cells/mL for 24 hours. The supernatant was collected, filtered through 0.22 μm filters, and immediately stored at −80°C.

Immunofluorescent Staining
The cells were fixed with 4% PFA for 15 min at 4°C, blocked in 1% BSA in PBS for 30 minutes at room temperature, incubated with anti-F4/80 antibody (eBiosciences, 14-4801-82) for 18 hours at 4°C, and incubated with an anti-rat IgG antibody (Thermo, Cat#A21210) and DAPI (1 μg/mL) for 30 minutes at 4°C. The stained cells were imaged using a Nikon Eclipse C2 confocal microscope (Nikon).

Isolation of bone marrow cells from mice
Bone marrow cells were aseptically harvested from euthanized 8–10 week-old C57BL/6 mice by flushing leg bones with RPMI, filtering through 70 μm cell strainers, and incubating in Red Blood Cell Lysing Buffer Hybri-Max™ (Sigma, R7757).

Bone marrow-derived macrophage viability assay (MTT)
Bone marrow cells were incubated with RPMI with 5% charcoal-stripped FBS, 50 ng/mL M-CSF (Peprotech, 315–02), and indicated concentrations of glutamine or conditioned media for 7 days. Cells were incubated with media containing thiazolyl blue tetrazolium bromide solution (Sigma, M2128) for two hours at 37°C. After the solution was removed, the blue crystalline precipitate in each well was dissolved in DMSO. Visible absorbance at 560 nm was quantified using a microplate reader.

Glutamine measurements
Glutamine concentrations in conditioned media were measured using a Glutamine/Glutamate-Glo Assay kit (Promega, J7021) following the manufacturer’s instructions.

Quantitative PCR
Total RNA was extracted using the RNeasy RNA Purification Kit (Qiagen, 75144) following the manufacturer’s protocol. cDNA was generated with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814). Quantitative PCR analyses were conducted on a Bio-Rad CFX96 system using TaqMan gene expression assays (Thermo Fisher Scientific): Nos2 (Mm00440502_m1), Il10 (Mm01288386_m1), Arg1 (Mm00475988_m1), Il12b (Mm01288989_m1), and Ppia (Mm02342430_g1).

Statistical analysis
Student’s t-test, one-sample t-test, or One-Way ANOVA was performed to compare independent sample groups. Excel (Microsoft) and Prism (GraphPad) were used for analysis. RNA-sequencing data from our prior publication comparing LLC cells with and without ectopic β3 expression were re-analyzed to identify differentially expressed genes between LLC-EV and +β3 cells associated with macrophage recruitment (Table 1).17

In vivo Mouse Tumor Experiments

Study approval
All experiments involving mice were conducted in accordance with a protocol approved by the UC San Diego Institutional Animal Care and Use Committee. All studies are in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Lung orthotopic xenograft model
HCC827 cells with and without integrin β3 ectopic expression (5 × 106 cells in 50 μL of PBS) were injected into the lungs of athymic nude mice (8–10-week-old) from Charles River Laboratories. Three months after the injection, the lungs with tumors were fixed in 10% formalin.

Lung orthotopic allograft model
LLC cells with and without integrin β3 ectopic expression (1 × 106 cells in 50 μL of PBS) were injected into the lungs of C57BL/6 mice (8–10-week-old) from Charles River Laboratories. Three months after the injection, the lungs with tumors were fixed in 10% formalin.

Genetically engineered NSCLC with integrin β3 knockout
Cc10-CreER; LSL-KrasG12D and Cc10-CreER; LSL-KrasG12D; Itgb3fl/fl (β3KO) mice were gifts from Dr. David Cheresh (San Diego, USA). The mice were treated weekly with naphthalene after 5 days of tamoxifen administration. At the end of the study, lungs were fixed in 10% formalin.

Tumor Initiation Assay
LLC cells with and without integrin β3 ectopic expression were injected subcutaneously into C57BL/6 mice (8–10-week-old) from Charles River Laboratories (5 groups: 1 × 103, 2 × 104, 5 × 104, 1 × 105, 1 × 106 cells in 100 μL of PBS). The mice were randomly divided into two groups: control (200 μL/mouse, i.p., twice per week, ClodronateLiposome.com, P-050) or clodronate liposome (200 μL/mouse, i.p., twice per week, ClodronateLiposome.com, C-030). Tumor take was monitored.

Data Availability
The data generated in this study are available upon request from the corresponding author.

Results

Integrin αvβ3 expression on cancer cells is necessary and sufficient to enrich for TAMs
Given the association between cancer cell integrin αvβ3 expression and TAM accumulation,15 we first asked if integrin αvβ3 is necessary and/or sufficient for increased TAMs in the microenvironment. To assess sufficiency, we compared macrophage abundance in orthotopic murine NSCLC model (LLC allografts) with or without ectopic integrin αvβ3 expression. Immunohistochemical (IHC) analysis revealed that αvβ3+ tumors contained significantly more F4/80+ macrophages compared to αvβ3- tumors (Fig. 1A), suggesting that tumor cell expression of integrin αvβ3 is sufficient to increase TAM infiltration. Given the complex crosstalk between cancer cells and the immune microenvironment—including regulatory roles of T cells in TAM recruitment21—we next asked whether the observed TAM enrichment was T cell dependent. To address this, we used an orthotopic xenograft model with HCC827 cells (human NSCLC) in athymic nude mice, which lack T cells. Ectopic expression of integrin αvβ3 in HCC827 cells led to significantly higher numbers of TAMs compared to control tumors (Fig. 1B), suggesting that integrin αvβ3-mediated TAM accumulation occurs independently of T cells.
To determine whether integrin αvβ3 expression is necessary for TAM infiltration, we used a naphthalene-induced lung cancer model in Cc10-CreER; LSL-KrasG12D mice with or without conditional deletion of integrin β3 (Itgb3fl/fl). In this model, naphthalene treatment induces lung adenocarcinoma in Cc10+ epithelial cells, along with upregulation of integrin αvβ3. Deletion of Itgb3 significantly reduced tumor burden (Fig. 1C), and IHC staining showed a marked decrease in F4/80+ macrophages in β3-knockout tumors (Fig. 1C). Together, these findings demonstrate that integrin αvβ3 expression on cancer cells is both necessary and sufficient to drive TAM enrichment in NSCLC.

Integrin αvβ3+ NSCLC cells secrete glutamine to increase TAMs
Cancer cells actively manipulate the immune microenvironment to their advantage by secreting factors such as cytokines, metabolites, and nucleotides.10–13 To investigate whether secreted factors from integrin αvβ3-expressing tumor cells enhance macrophage viability, we collected conditioned media from murine NSCLC LLC cells with or without ectopic integrin αvβ3 expression and used it to culture murine bone marrow cells (Fig. 2A). We then assessed macrophage viability and differentiation using a viability assay and immunocytochemistry for the macrophage marker F4/80. Bone marrow cells treated with conditioned media from αvβ3+ cells yielded a greater number of F4/80+ macrophages compared to those treated with media from αvβ3− cells (Fig. 2A), while conditioned media from αvβ3+ cells did not change macrophage phenotype (Fig. S1A). These findings suggest that integrin αvβ3-expressing tumor cells secrete factors that promote macrophage differentiation from bone marrow precursors, enhance macrophage proliferation, and/or improve macrophage survival.
To identify secreted factors that promote macrophage infiltration, we first screened integrin αvβ3− and αvβ3+ LLC cells for cytokines and growth factors known to regulate macrophage recruitment or proliferation (CCL2, CCL5, CSF1, CSF2, IL-1B, IL-4, IL-6, IL-13, tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF)-A) in our previously published RNA-sequencing dataset from LLC-EV and +β3 cells.17 Differential expression analysis revealed a statistically significant upregulation of Ccl2 in β3+ LLC cells (>2-fold, P < 0.05; Table 1). However, CCL2 protein levels were not elevated in conditioned media from LLC+β3 cells (Fig. S1B). We next considered whether metabolites were responsible for the increase in macrophages. We previously reported that integrin αvβ3 upregulates glutamine metabolism in NSCLC cells.17 Given that glutamine is essential for alveolar macrophage proliferation,22 we next asked whether αvβ3+ NSCLC cells secrete glutamine. Indeed, in conditioned media from LLC+β3 cells, we detected approximately 300 μM glutamine, indicating that ~200 μM glutamine was secreted by LLC+β3 cells, given that the media was supplemented with 100 μM glutamine (Fig. 2C). In contrast, no glutamine was detected in conditioned media from LLC-EV cells (Fig. 2C), suggesting increased glutamine secretion by integrin αvβ3-expressing cells. To determine whether glutamine promotes macrophage emergence from bone marrow cells, we cultured murine bone marrow cells with increasing concentrations of glutamine, including the physiological level found in human serum (~0.3 mM). Viability assays revealed a dose-dependent increase in macrophage differentiation from bone marrow cells derived from both female and male mice (Fig. 2D).

TAMs are essential for integrin αvβ3-mediated enhanced tumorigenicity.
Integrin αvβ3 promotes cancer stemness, including enhanced tumor-initiating capacity, across multiple solid tumors, including NSCLC.15–18,23 Given that TAMs support tumor development and initiation—and that integrin αvβ3 expression on cancer cells was both necessary and sufficient to increase TAMs in the tumor microenvironment (Fig. 1)—we asked whether macrophage recruitment contributes to the enhanced tumor-initiating capacity of αvβ3+ cells. To test this, we performed an in vivo tumor initiation assay (limiting dilution assay) using the LLC (murine NSCLC) allograft model with or without ectopic expression of integrin αvβ3, and with or without macrophage depletion using clodronate liposomes. As expected, αvβ3+ cells exhibited greater tumor-initiating capacity compared to αvβ3− cells; however, this advantage was significantly reduced when macrophages were depleted (Fig. 3A and B). Interestingly, in mice injected with high numbers of tumor cells—where tumor initiation rates were comparable—macrophage depletion did not alter overall tumor growth. In contrast, αvβ3+ xenografts exhibited markedly accelerated growth, suggesting that the mechanisms governing tumor initiation and subsequent growth are distinct (Fig. 3A). Together, these results indicate that integrin αvβ3+ tumors, at least in part, depend on macrophages to support their tumor-initiating capacity but not tumor growth.

Discussion
Tumor-associated macrophages (TAMs) play critical roles in cancer progression by promoting angiogenesis, immune suppression, tumor growth, and metastasis.1–3,24 While cancer stem-like cells can drive TAM recruitment,25,26 TAMs, in turn, reinforce stem-like phenotypes in cancer cells by secreting growth factors and cytokines. However, the mechanisms by which NSCLC cells promote TAM infiltration into the tumor microenvironment remain incompletely understood. In this study, we identify integrin αvβ3 expression on NSCLC cells as both necessary and sufficient to drive TAM accumulation in vivo (Fig. 1). We further show that integrin αvβ3+ NSCLC cells secrete glutamine, which enhances macrophage survival and/or differentiation (Fig. 2), and that TAMs are required for the elevated tumor-initiating capacity of αvβ3+ tumors (Fig. 3). Together, these findings support a model in which cancer cell-intrinsic integrin αvβ3 programs the immune microenvironment to promote tumor initiation.
Our results demonstrate that αvβ3 expression alone is sufficient to increase TAM infiltration in orthotopic tumor models, and that this effect occurs independently of T cells, as evidenced by the increase in TAMs in αvβ3+ tumors implanted in athymic nude mice (Fig. 1B). This suggests that tumor-intrinsic αvβ3 signaling directly regulates TAM accumulation, independent of adaptive immunity. In support of this, conditioned media from αvβ3+ NSCLC cells increased the number of F4/80+ cells in cultured murine bone marrow, suggesting enhanced differentiation, proliferation, and/or survival of macrophages due to tumor-derived soluble factors (Fig. 2A). While previous studies have correlated integrin αvβ3 expression with aggressive tumor phenotypes and poor prognosis,16–18 our work extends this understanding by uncovering a direct link between tumor αvβ3 expression and TAM dynamics in NSCLC.
Mechanistically, we found that αvβ3+ NSCLC cells secrete elevated levels of glutamine, rather than canonical TAM-regulatory cytokines, to support macrophage accumulation (Table 1, Fig. 2B–C). This finding aligns with a recent study in the lungs, which presents that glutamine is critical for the proliferation of alveolar macrophages.22 Glutamine is also a critical nutrient for NSCLC cell survival under metabolic stress and in drug-resistant states. Our findings suggest that previously reported enhanced glutamine metabolism by integrin αvβ3 in NSCLC cells is not only to support their own anabolic needs but also to foster a macrophage-rich, pro-tumorigenic immune niche. Although we did not observe a significant increase in cytokine or growth factor secretion from αvβ3+ NSCLC cells in vitro, it remains possible that these factors, acting collectively, promote TAM infiltration in vivo. For example, cytokines may recruit macrophages to the tumor site, while elevated glutamine secretion could enhance the survival or proliferation of the recruited macrophages.
Importantly, we demonstrate that TAMs are functionally required for the enhanced tumor-initiating capacity of αvβ3+ cells. Depleting macrophages with clodronate liposomes attenuated the tumorigenic advantage conferred by integrin αvβ3 (Fig. 3), suggesting that TAMs actively contribute to αvβ3-driven tumor initiation. This finding is consistent with emerging evidence that TAMs support stem-like properties in cancer cells through cytokine production, matrix remodeling, and immunosuppressive functions.1–7
In conclusion, these findings show that integrin αvβ3+ NSCLC cells secrete glutamine, thereby promoting TAM accumulation in the tumor microenvironment. While integrin αvβ3 has long been recognized as a cancer stem-like cell marker, which drives metastasis and drug resistance, this study provides, to our knowledge, the first evidence that αvβ3+ cancer cells directly shape the tumor immune landscape. These results add to the growing understanding of how NSCLC cells exploit the tumor microenvironment, highlighting one mechanism—mediated by integrin αvβ3—by which TAMs are recruited.

Supplementary Material
Supplementary Figures