Tumor Microenvironment In Mouse Model Research Articles

Abstract NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer

Abstract NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer

Anlotinib Enhances the Antitumor Activity of High-Dose Irradiation Combined with Anti-PD-L1 by Potentiating the Tumor Immune Microenvironment in Murine Lung Cancer

<h3>Purpose/Objective(s)</h3> Radioimmunotherapy has become one of the most promising strategies for cancer treatment. Preclinical and clinical studies have demonstrated that antiangiogenic therapy can improve the efficacy of immunotherapy and sensitize radiotherapy through a variety of mechanisms. However, it is undefined whether angiogenesis inhibitors can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of anlotinib (AL3818) on the combination of radiotherapy and immune checkpoint inhibitors in Lewis lung carcinoma mouse. <h3>Materials/Methods</h3> C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control. Immune response and immunophenotyping including the quantification and activation were determined by flow cytometry, multiplex immunofluorescence and multiplex immunoassay. <h3>Results</h3> Triple therapy (radiotherapy combined with anti-PD-L1 and anlotinib) increased tumor-infiltrating lymphocytes and reversed the immunosuppressive effect of radiation on the tumor microenvironment in mouse model. Compared with radioimmunotherapy, the addition of anlotinib also boosted the infiltration of CD8⁺ T cells and M1 cells, and caused a decrease in the number of MDSCs and M2 cells in mice. The levels of IFN-gamma, IL-18 were the highest in the triple therapy group, while the levels of IL-23, IL-13, IL-1 beta, IL-2, IL-6, IL-10 and Arg-1 were significantly reduced. NF-κB, MAPK and AKT pathways were down regulated in triple therapy compared with radioimmunotherapy. Thus, the tumor immune microenvironment was significantly improved. As a consequence, triple therapy displayed greater benefit in antitumor efficacy. <h3>Conclusion</h3> Our findings indicate that anlotinib might be a potential synergistic treatment for radioimmunotherapy to achieve better antitumor efficacy in NSCLC patients by potentiating the tumor immune microenvironment.

C3aR Signaling Inhibits NK-cell Infiltration into the Tumor Microenvironment in Mouse Models.

Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells, suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells, but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration, we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells, we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1, which led to a high-affinity LFA-1 conformation, decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME, through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1, represent a new strategy to improve the efficiency of immunotherapy for cancer treatment.

Anlotinib Enhances the Antitumor Activity of High-Dose Irradiation Combined with Anti-PD-L1 by Potentiating the Tumor Immune Microenvironment in Murine Lung Cancer.

Background Radioimmunotherapy has become one of the most promising strategies for cancer treatment. Preclinical and clinical studies have demonstrated that antiangiogenic therapy can improve the efficacy of immunotherapy and sensitize radiotherapy through a variety of mechanisms. However, it is undefined whether angiogenesis inhibitors can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of anlotinib (AL3818) on the combination of radiotherapy and immune checkpoint inhibitors in Lewis lung carcinoma mouse. Methods C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control. Immune response and immunophenotyping including the quantification and activation were determined by flow cytometry, multiplex immunofluorescence, and multiplex immunoassay. Results Triple therapy (radiotherapy combined with anti-PD-L1 and anlotinib) increased tumor-infiltrating lymphocytes and reversed the immunosuppressive effect of radiation on the tumor microenvironment in mouse model. Compared with radioimmunotherapy, the addition of anlotinib also boosted the infiltration of CD8+ T cells and M1 cells and caused a decrease in the number of MDSCs and M2 cells in mice. The levels of IFN-gamma and IL-18 were the highest in the triple therapy group, while the levels of IL-23, IL-13, IL-1 beta, IL-2, IL-6, IL-10, and Arg-1 were significantly reduced. NF-κB, MAPK, and AKT pathways were downregulated in triple therapy compared with radioimmunotherapy. Thus, the tumor immune microenvironment was significantly improved. As a consequence, triple therapy displayed greater benefit in antitumor efficacy. Conclusion Our findings indicate that anlotinib might be a potential synergistic treatment for radioimmunotherapy to achieve better antitumor efficacy in NSCLC patients by potentiating the tumor immune microenvironment.

Development of a Nanoscale Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agent That Measures pH.

AcidoCEST MRI can measure the extracellular pH (pHe) of the tumor microenvironment in mouse models of human cancers and in patients who have cancer. However, chemical exchange saturation transfer (CEST) is an insensitive magnetic resonance imaging (MRI) contrast mechanism, requiring a high concentration of small-molecule agent to be delivered to the tumor. Herein, we developed a nanoscale CEST agent that can measure pH using acidoCEST MRI, which may decrease the requirement for high delivery concentrations of agent. We also developed a monomer agent for comparison to the polymer. After optimizing CEST experimental conditions, we determined that the polymer agent could be used during acidoCEST MRI studies at 125-fold and 488-fold lower concentration than the monomer agent and iopamidol, respectively. We also determined that both agents can measure pH with negligible dependence on temperature. However, pH measurements with both agents were dependent on concentration, which may be due to concentration-dependent changes in hydrogen bonding and/or steric hindrance. We performed in vivo acidoCEST MRI studies using the three agents to study a xenograft MDA-MB-231 model of mammary carcinoma. The tumor pHe measurements were 6.33 ± 0.12, 6.70 ± 0.15, and 6.85 ± 0.15 units with iopamidol, the monomer agent, and polymer agent, respectively. The higher pHe measurements with the monomer and polymer agents were attributed to the concentration dependence of these agents. This study demonstrated that nanoscale agents have merit for CEST MRI studies, but consideration should be given to the dependence of CEST contrast on the concentration of these agents.

Abstract 2768: A novel non-terminal tumor sampling procedure using fine needle aspiration supports biomarker discovery

Abstract Immunotherapy is part of the standard of care for oncology however, durable objective responses remain limited to a subset of patients. As such there is a crucial need to identify biomarkers that can predict/enrich for treatment response. So far, the majority of proposed biomarkers consist of features of the tumor microenvironment (TME). However, in preclinical mouse models, the collection of tumor tissue for this type of analysis is a terminal procedure obviating the ability to directly link potential biomarkers to response/outcome following treatment. Therefore, we have developed and validated a novel non-terminal tumor sampling method to biopsy the TME in mouse models based on fine needle aspiration. We show that this technique enables repeated in-life sampling of subcutaneous flank tumors without impacting tumor growth or animal welfare. This method yields sufficient sample for flow cytometric analysis of tumor-infiltrating immune cells and we demonstrate that it is able to recapitulate results obtained with the current methodology of whole tumor analysis. We propose that this method represents a simple, fast, minimally invasive technique to enable analysis of the TME, reduction in the number of animals used for preclinical oncology studies and refinement of pharmacodynamic analysis. Additionally, its unique ability to provide longitudinal TME sampling can support investigation of biomarkers of response to treatment. Citation Format: Suzanne I. Sitnikova, Sophie Munnings-Tomes, Stacy R. Kentner, Kathy Mulgrew, Judit Espana-Agusti, Tianhui Zhang, Kristina M. Ilieva, Hormas M. Ghadially, Matthew J. Robinson, Robert W. Wilkinson, Simon J. Dovedi. A novel non-terminal tumor sampling procedure using fine needle aspiration supports biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2768.

Novel non-terminal tumor sampling procedure using fine needle aspiration supports immuno-oncology biomarker discovery in preclinical mouse models

BackgroundImmuno-oncology therapies are now part of the standard of care for cancer in many indications. However, durable objective responses remain limited to a subset of patients. As such, there is...

Aptamers against mouse and human tumor-infiltrating myeloid cells as reagents for targeted chemotherapy.

Local delivery of anticancer agents has the potential to maximize treatment efficacy and minimize the acute and long-term systemic toxicities. Here, we used unsupervised systematic evolution of ligands by exponential enrichment to identify four RNA aptamers that specifically recognized mouse and human myeloid cells infiltrating tumors but not their peripheral or circulating counterparts in multiple mouse models and from patients with head and neck squamous cell carcinoma (HNSCC). The use of these aptamers conjugated to doxorubicin enhanced the accumulation and bystander release of the chemotherapeutic drug in both primary and metastatic tumor sites in breast and fibrosarcoma mouse models. In the 4T1 mammary carcinoma model, these doxorubicin-conjugated aptamers outperformed Doxil, the first clinically approved highly optimized nanoparticle for targeted chemotherapy, promoting tumor regression after just three administrations with no detected changes in weight loss or blood chemistry. These RNA aptamers recognized tumor infiltrating myeloid cells in a variety of mouse tumors in vivo and from human HNSCC ex vivo. This work suggests the use of RNA aptamers for the detection of myeloid-derived suppressor cells in humans and for a targeted delivery of chemotherapy to the tumor microenvironment in multiple malignancies.

Effect of general anesthetics on the tumor micro-environment in mouse models of breast cancers of spontaneous metastasis.

e15503 Background: Metastatic breast cancer is a pressing health concern worldwide. Various treatments have been developed but no significant long-term changes in overall survival are observed. Therefore, there is a demand to improve current therapies to treat this disease. Surgical resection of the primary tumors is essential in the treatment. However, accumulating evidence alludes to a role for volatile anesthetics which are used during the surgery in metastatic tumor development, but the mechanism remains largely unknown. We have shown anesthetics exert different effects on lung metastasis in mouse models of breast cancers. This study analyses the effect of general anesthetics in lung microenvironment associated with the increased metastases. Methods: Balb/c mice and NOD-SCID mice were orthotopically implanted with 4T1 cells and MDA-MB-231 cells respectively, in the mammary fat pad to generate primary tumors. Mice were subjected to the tested anesthetic during implantation and/or before and after surgery. Surgical dissection of primary tumor was performed under anesthesia with sevoflurane or an intravenous anesthetic propofol. Survival curve was constructed and analysed. Mice were euthanized to harvest tissues for histology and cell analysis. Results: As we previously reported, surgical dissection of primary tumor in mice under anesthesia with sevoflurane led to significantly more lung metastasis than with propofol in both syngeneic murine 4T1 and xenograft human MDA-MB-231 breast cancer models. Sevoflurane was associated with increased IL6(Li, Huang, &amp; Lin, 2020). Here we show that anesthesia with sevoflurane resulted in changes of stroma composition in the lung, which was reversed by IL6 pathway interruption. Conclusions: Those results contribute to our understanding of effects of sevoflurane on cancer metastasis and suggest a potential therapeutic approach to overcome the risk of general anesthesia. Li, R., Huang, Y., &amp; Lin, J. (2020). Distinct effects of general anesthetics on lung metastasis mediated by IL-6/JAK/STAT3 pathway in mouse models. Nat Commun, 11, 642.

PGE2-HIF1α reciprocal induction regulates migration, phenotypic alteration and immunosuppressive capacity of macrophages in tumor microenvironment

BackgroundVery little is known about the role inflammation and mechanism(s) that enables the tumor to evade host's anti-tumor immune function during very initial days of tumor establishment. Our study focuses on the immune response and local inflammation specially the pro-inflammatory and immune modifier components that are responsible for tumor-induced immune-suppression, tumor-associated macrophages (TAM) at tumor microenvironment in mouse model from very early to late phase of tumor progression. Methods1 × 105 Ascites tumor, EAC in Swiss albino or Sarcoma-180 (S-180) in Balb c mice strain were inoculated intra-peritonially and grouped into Control (0 day or no tumor), initial phase (3 day tumor), early (7 Day), Late (14 day) and terminal (21 day tumor) sets. T cell activity, tumor niche macrophage, inflammatory signatures were studied using Confocal microscopy, flowcytometry, ELISA, q-RT PCR and Western blot. ResultsWe observed increased T cell infiltration at a very early stage of tumorigenesis in the tumor site with elevated percentage of activated/memory T cells. But increased cellular death and functional suppression of tumor site T cells during final stages. We observed increased infiltration of TAMs with skewed M2 phenotype. Increased chemokine receptor expression could be noted on these TAMs. Using HIF-1α inhibitor and prostaglandin receptor antagonists we demonstrated crucial role of these factor in functional alteration in TAMs. HIF-1α inhibition and also by prostaglandin receptor inhibition reduced signature pro-inflammatory gene expression, migration of macrophages and T cell suppression capacity of TAMs. We also demonstrated that PGE2 can induce HIF-1α activation in relatively less hypoxic microenvironment during early stages of tumor. ConclusionAltogether, these findings strongly suggest link between prostaglandin mediated early HIF-1α activation and subsequent hypoxia induced HIF-1α activation that further enhances prostaglandin synthesis driving the recruitment and functional alteration of tumor site macrophages.

Abstract A77: Differential recruitment of myeloid derived immune cells and fibroblasts to the thyroid tumor microenvironment in mouse models of papillary and follicular thyroid cancer

Abstract Thyroid cancer is the most common endocrine malignancy, and its incidence has steadily increased worldwide over the last 30 years. Genetic alterations leading to the activation of the MAPK signaling pathway are crucial for the initiation and progression of thyroid cancer, as evidenced by the high frequency of activating mutations in BRAF, HRAS, NRAS, RET and TRK. Mutations in BRAF are observed in ~45% of papillary thyroid cancers (PTCs), while follicular thyroid cancer (FTC) is often associated with mutations in RAS. We have shown that thyroid specific expression of HrasG12V and Pten inactivation leads to the development of FTCs and poorly differentiated thyroid cancer (PDTC) in HrasG12V/Ptenhom/TPO-cre mice while thyroid specific expression of BrafV600E and loss of Pten leads to the rapid development of PTC and PDTC in BrafV600E/Ptenhom/TPO-cre mice with complete lethality by weaning. We sought to determine whether activation of Braf or Hras resulted in the differential recruitment of stromal cells to the thyroid tumor microenvironment in HrasG12V/Ptenhom/TPO-cre and BrafV600E/Ptenhom/TPO-cre mice. FACS analysis revealed a significant increase in the overall amount of CD45+ cells in Hras (60.95±5.28%) versus Braf (18.35±1.3%) driven murine thyroid tumors. However, we found that the population of CD45+ cells in Braf driven tumors was comprised of a higher percentage of CD11b+ myeloid cells (62.1±1.3%) in comparison to Hras (37.0±5.5%) driven tumors. We next measured levels of cytokine expression in tumor cell lines derived from BrafV600E/Ptenhom/TPO-cre and HrasG12V/Ptenhom/TPO-cre mice. Braf driven tumor cells expressed higher levels of GM-CSF in comparison to Hras driven tumor cells, which we hypothesize contributes to the larger myeloid compartment observed in BrafV600E/Ptenhom/TPO-cre thyroid tumors. Studies are ongoing to further characterize the immune cell populations in these tumors. FACS analysis also revealed an increase in CD45-CD326-CD140α+ fibroblast infiltrate in Braf versus Hras driven thyroid tumors (68.4±0.424% versus 9.8±4.27%, p&amp;lt;.0001). Fibroblasts are a major source of type I collagen (Col1a1), which has been linked to the immunosuppression of tumor infiltrating leukocytes via inhibition of Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). Therefore, we hypothesized that the increase in fibroblast infiltrate observed in Braf driven thyroid tumors would result in a subsequent increase in Col1a1 production, thus potentially leading to a more immunosuppressive thyroid tumor microenvironment in PTC. Indeed, levels of Col1a1 gene expression were significantly upregulated an average of 80 fold (p&amp;lt;.0001) in Braf versus Hras driven thyroid tumors, with no significant difference in Col1a1 expression between Hras driven tumors and WT thyroid controls. Histopathological analysis via Picrosirius red staining confirmed higher levels of total and fibrillar collagen in Braf versus Hras driven tumors. In addition, microarray analysis of human thyroid tumors revealed that Col1a1 expression levels are highest in PTCs, particularly in those with mutations in BRAF, and lowest in FTCs and follicular variant PTC, which are both associated with a high RAS mutation rate. Because FTCs and PTCs have distinct pathological features and differ in prognosis and responses to therapy, we aim to translate these findings to the discovery of more personalized diagnostic and treatment strategies that are targeted toward the thyroid tumor microenvironment. Citation Format: Lee Ann King, Sergey Novitsky, Julio Ricarte-Filho, Philip Owens, Aime T. Franco. Differential recruitment of myeloid derived immune cells and fibroblasts to the thyroid tumor microenvironment in mouse models of papillary and follicular thyroid cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A77.

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

Tumor Microenvironment–Dependent 18F-FDG, 18F-Fluorothymidine, and 18F-Misonidazole Uptake: A Pilot Study in Mouse Models of Human Non–Small Cell Lung Cancer

(18)F-FDG, (18)F-fluorothymidine, and (18)F-misonidazole PET scans have emerged as important clinical tools in the management of cancer; however, none of them have demonstrated conclusive superiority. The aim of this study was to compare the intratumoral accumulation of (18)F-FDG, (18)F-fluorothymidine, and (18)F-misonidazole and relate this to specific components of the tumor microenvironment in mouse models of human non-small cell lung cancer (NSCLC). We used NSCLC A549 and HTB177 cells to generate subcutaneous and peritoneal xenografts in nude mice. Animals were coinjected with a PET radiotracer, pimonidazole (hypoxia marker), and bromodeoxyuridine (proliferation marker) intravenously 1 h before animal euthanasia. Tumor perfusion was assessed by Hoechst 33342 injection, given 1 min before sacrifice. The intratumoral distribution of PET radiotracers was visualized by digital autoradiography and related to microscopic visualization of proliferation, hypoxia, perfusion, stroma, and necrosis. NSCLC xenografts had complex structures with intermingled regions of viable cancer cells, stroma, and necrosis. Cancer cells were either well oxygenated (staining negatively for pimonidazole) and highly proliferative (staining positively for bromodeoxyuridine) or hypoxic (pimonidazole-positive) and noncycling (little bromodeoxyuridine). Hypoxic cancer cells with a low proliferation rate had high(18)F-FDG and (18)F-misonidazole uptake but low (18)F-fluorothymidine accumulation. Well-oxygenated cancer cells with a high proliferation rate accumulated a high level of (18)F-fluorothymidine but low (18)F-FDG and(18)F-misonidazole. Tumor stroma and necrotic zones were always associated with low (18)F-FDG, (18)F-misonidazole, and (18)F-fluorothymidine activity. In NSCLC A549 and HTB177 subcutaneously or intraperitoneally growing xenografts, (18)F-fluorothymidine accumulates in well-oxygenated and proliferative cancer cells, whereas (18)F-misonidazole and (18)F-FDG accumulate mostly in poorly proliferative and hypoxic cancer cells. (18)F-FDG and (18)F-misonidazole display similar intratumoral distribution patterns, and both mutually exclude (18)F-fluorothymidine.

Abstract 2439: Molecular imaging of spatial and temporal heterogeneity of tumor micro-environment in mouse models of non-small cell lung cancer macroscopic xenografts and micro-metastases

Abstract 18F-fluoro-2-deoxyglucose (18F-FDG, imaging glucose metabolism), 18F-fluorothymidine (18F-FLT, cell proliferation) and 18F-misonidazole (18F-FMISO, hypoxia) PET scans have emerged as important clinical tools for management of cancer. The objective of this study was to spatially and temporally visualize tumor microenvironment of tumor hypoxia, proliferation and glucose metabolism using 18F-FDG, 18F-FLT and 18F-FMISO digital autoradiography and microPET comparing with histological findings. Methods: We used human non-small cell lung cancer (NSCLC) A549 and HTB177 cells to generate subcutaneous and peritoneal metastases in nude mice. Animals were coinjected with the mixture of one PET radiotracer, pimonidazole (hypoxia marker) and bromodeoxyuridine (proliferation marker) intravenously 1 hour before animal euthanasia. The intratumoral distributions of radiotracers were visualized by digital autoradiography (DAR) and related to microscopic visualization of cellular proliferation, tumor hypoxia, stroma and necrosis. Serial microPET scans (day 1to day5) were also performed in the same animals to investigate change in glucose metabolism (using 18F-FDG), proliferation (18F-FLT) and hypoxia (18F-FMISO). Results: NSCLC microenvironment was complex and highly heterogeneous: xenografts had complex structures with intermingled regions of well oxygenated (negative pimonidazole) and highly proliferative (positive bromodeoxyuridine) cancer cells, hypoxic (positive pimonidazole) and low proliferation (little bromodeoxyuridine) cancer cells stroma and necrosis. Hypoxic cancer cells had high18F-FDG and 18F-FMISO but low 18F-FLT accumulation, indicating increased glucose metabolism is not a common feature of cancer cells but only hypoxic ones. Well oxygenated cancer cells with high proliferation rate accumulated high level of 18F-FLT, but low 18F-FDG and18F-FMISO. Stroma and necrotic zones always associated with low activity in all radiotracers we tested. MicroPET scans revealed apparent change in intratumor distribution of 18F-FLT, 18F-FDG as well as 18F-FMISO in as short as ∼48 hrs interval, indicating temporal heterogeneity of tumor microenvironment in term of proliferation, glucose metabolism and hypoxia. Conclusions: Both macroscopic xenografts and micrometastases of NSCLC have spatial heterogeneity of tumor microenvironment. Temporal change of tumor microenvironment did occur in a very short of interval of natural growth process in NSCLC. We have investigated to spatial and temporal behavior of heterogeneity of tumor microenvironment which is important for better understanding cancer biology and cancer management, if our findings in mice models are clinically applicable. Acknowledgement: This study was supported by Kentucky Lung Cancer Research Program Award to Dr. Xiao-Feng Li. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2439. doi:1538-7445.AM2012-2439