Lung Tumor Growth In Mice Research Articles

Abstract 1352: Computational design of a cyclic peptide that inhibits the CTLA4 in T cells and the growth of lung carcinoma in mice

Abstract Proteins involved in immune checkpoint pathways, such as CTLA4, PD-1, and PD-L1, have become important targets for cancer immunotherapy; however, the development of small molecule drugs targeting these pathways has proven difficult due to the nature of their protein-protein interfaces. Here, using a hierarchy of computational techniques, we designed a cyclic peptide that binds CTLA4 and follow this with experimental verification of binding and biological activity, using bio-layer interferometry, cell culture, and a lung cancer mouse model in immunocompetent mice. Beginning from a template excised from the x-ray structure of the CTLA4:B7-2 complex, we generate several peptide sequences using Rosetta, a protein modelling program. These peptides are cyclized head-to-tail to improve structural and proteolytic stability and screened using molecular dynamics simulation and MM-GBSA calculation. The standard binding free energies for shortlisted peptides are then calculated in explicit-solvent simulation using a rigorous multistep Binding Free Energy Estimator (BFEE). The most promising peptide, cyc-EIDTVLTPTGWVAKRYS (hereafter CTLA4-ip), yields the standard free energy -6.6 ± 3.5 kcal/mol, which corresponds to a dissociation constant of 15 μmol/L. The binding affinity of this peptide for CTLA4 is measured experimentally (31 ± 4 μmol/L) using bio-layer interferometry. Pharmacokinetics of CTLA4-ip in a cell culture with Lewis lung carcinoma (LLC) cells and in mice revealed that this peptide is significantly more stable in both cell culture and in mice as compared to a control non-cyclic scrambled-sequence. Treatment with CTLA4-ip inhibited cancer cell growth in a co-culture of LLC cells and LLC cell-antigen primed murine T cells. Intraperitoneal administration of the CTLA4-ip (10mg/kg/day, every other day totaling 4 doses) markedly inhibited lung tumor growth in mice with an orthotropic LLC allograft model. Efficacy of the tumor growth inhibition by the CTLA4-ip was similar to that by an anti-PD-L1 antibody (10mg/kg/day, every two days totaling 3 doses). These results strongly suggest that this novel CTLA4-ip works as an immune checkpoint inhibitor to CTLA4 and is usable for lung cancer treatment. This research was supported by Kansas State University Johnson Cancer Research center (MT), Midwest Biomedical Accelerator Consortium (MT, JC), National Cancer Institute MT, JC) and the National Science Foundation (JC). Citation Format: Ravindra Thakkar, Deepa Upreti, Susumu Ishiguro, Geraldine Magnin, Keshari Sudasinghe, Anabelle Hall, Sarah DeVader, Masaaki Tamura, Jeffrey Comer. Computational design of a cyclic peptide that inhibits the CTLA4 in T cells and the growth of lung carcinoma in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1352.

Expanding individualized therapeutic options via genoproteomics

Clinical next-generation sequencing (NGS) tests have enabled treatment recommendations for cancer patients with driver gene mutations. Targeted therapy options for patients without driver gene mutations are currently unavailable. Herein, we performed NGS and proteomics tests on 169 formalin-fixed paraffin-embedded (FFPE) samples of non-small cell lung cancers (NSCLC, 65), colorectal cancers (CRC, 61), thyroid carcinomas (THCA, 14), gastric cancers (GC, 2), gastrointestinal stromal tumors (GIST, 11), and malignant melanomas (MM, 6). Of the 169 samples, NGS detected 14 actionable mutated genes in 73 samples, providing treatment options for 43% of the patients. Proteomics identified 61 actionable clinical drug targets approved by the FDA or undergoing clinical trials in 122 samples, providing treatment options for 72% of the patients. In vivo experiments demonstrated that the Mitogen-Activated Protein Kinase (MEK) inhibitor could block lung tumor growth in mice with overexpression of Map2k1 protein. Therefore, protein overexpression is a potentially feasible indicator for guiding targeted therapies. Collectively, our analysis suggests that combining NGS and proteomics (genoproteomics) could expand the targeted treatment options to 85% of cancer patients.

Mouse pulmonary interstitial macrophages mediate the pro-tumorigenic effects of IL-9

Although IL-9 has potent anti-tumor activity in adoptive cell transfer therapy, some models suggest that it can promote tumor growth. Here, we show that IL-9 signaling is associated with poor outcomes in patients with various forms of lung cancer, and is required for lung tumor growth in multiple mouse models. CD4+ T cell-derived IL-9 promotes the expansion of both CD11c+ and CD11c− interstitial macrophage populations in lung tumor models. Mechanistically, the IL-9/macrophage axis requires arginase 1 (Arg1) to mediate tumor growth. Indeed, adoptive transfer of Arg1+ but not Arg1- lung macrophages to Il9r−/− mice promotes tumor growth. Moreover, targeting IL-9 signaling using macrophage-specific nanoparticles restricts lung tumor growth in mice. Lastly, elevated expression of IL-9R and Arg1 in tumor lesions is associated with poor prognosis in lung cancer patients. Thus, our study suggests the IL-9/macrophage/Arg1 axis is a potential therapeutic target for lung cancer therapy.

Ninjurin1 drives lung tumor formation and progression by potentiating Wnt/\u03b2-Catenin signaling through Frizzled2-LRP6 assembly

BackgroundCancer stem-like cells (CSCs) play a pivotal role in lung tumor formation and progression. Nerve injury-induced protein 1 (Ninjurin1, Ninj1) has been implicated in lung cancer; however, the pathological role of Ninj1 in the context of lung tumorigenesis remains largely unknown.MethodsThe role of Ninj1 in the survival of non-small cell lung cancer (NSCLC) CSCs within microenvironments exhibiting hazardous conditions was assessed by utilizing patient tissues and transgenic mouse models where Ninj1 repression and oncogenic KrasG12D/+ or carcinogen-induced genetic changes were induced in putative pulmonary stem cells (SCs). Additionally, NSCLC cell lines and primary cultures of patient-derived tumors, particularly Ninj1high and Ninj1low subpopulations and those with gain- or loss-of-Ninj1 expression, and also publicly available data were all used to assess the role of Ninj1 in lung tumorigenesis.ResultsNinj1 expression is elevated in various human NSCLC cell lines and tumors, and elevated expression of this protein can serve as a biomarker for poor prognosis in patients with NSCLC. Elevated Ninj1 expression in pulmonary SCs with oncogenic changes promotes lung tumor growth in mice. Ninj1high subpopulations within NSCLC cell lines, patient-derived tumors, and NSCLC cells with gain-of-Ninj1 expression exhibited CSC-associated phenotypes and significantly enhanced survival capacities in vitro and in vivo in the presence of various cell death inducers. Mechanistically, Ninj1 forms an assembly with lipoprotein receptor-related protein 6 (LRP6) through its extracellular N-terminal domain and recruits Frizzled2 (FZD2) and various downstream signaling mediators, ultimately resulting in transcriptional upregulation of target genes of the LRP6/β-catenin signaling pathway.ConclusionsNinj1 may act as a driver of lung tumor formation and progression by protecting NSCLC CSCs from hostile microenvironments through ligand-independent activation of LRP6/β-catenin signaling.

Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis

Pyroptosis, a type of programmed cell death (PCD), is characterized by cell swelling with bubbles, and the release of inflammatory cell cytokines. Cucurbitacin B (CuB), extracted from muskmelon pedicel, is a natural bioactive product that could effectively exert anti-tumor activities in lung cancer. However, the exact molecular mechanisms and the direct targets of CuB in non-small cell lung cancer (NSCLC) remain to be discovered. Here, we firstly found that CuB exerted an anti-tumor effect via pyroptosis in NSCLC cells and NSCLC mice models. Next, based on the molecular docking and cellular thermal shift assay (CETSA), we identified that CuB directly bound to Toll-like receptor 4 (TLR4) to activate the NLRP3 inflammasome, which further caused the separation of N- and C-terminals of Gasdermin D (GSDMD) to execute pyroptosis. Moreover, CuB enhanced the mitochondrial reactive oxygen species (ROS), mitochondrial membrane protein Tom20 accumulation, and cytosolic calcium (Ca2+) release, leading to pyroptosis in NSCLC cells. Silencing of TLR4 inhibited CuB-induced pyroptosis and decreased the level of ROS and Ca2+ in A549 cells. In vivo study showed that CuB treatment suppressed lung tumor growth in mice via pyroptosis without dose-dependent manner, and CuB at 0.75 mg/kg had a better anti-tumor effect compared to the Gefitinib group. Taken together, our findings revealed the mechanisms and targets of CuB triggering pyroptosis in NSCLC, thus supporting the notion of developing CuB as a promising therapeutic agent for NSCLC.

Recombinant Adeno-associated Virus 9-mediated Expression of Kallistatin Suppresses Lung Tumor Growth in Mice.

Lung cancer remains the most common cause of cancer-related deaths in China and worldwide. Traditional surgery and chemotherapy do not offer an effective cure, although gene therapy may be a promising future alternative. Kallistatin (Kal) is an endogenous inhibitor of angiogenesis and tumorigenesis. Recombinant adeno-associated virus (rAAV) is considered the most promising vector for gene therapy of many diseases due to persistent and long-term transgenic expression. The aim of this study was to investigate whether rAAV9-Kal inhibited NCI-H446 subcutaneous xenograft tumor growth in mice. The subcutaneous xenograft mode was induced by subcutaneous injection of 2×107 H446 cells into the dorsal skin of BALB/c nude mice. The mice were administered with ssrAAV9-Kal (single- stranded rAAV9) or dsrAAV9-Kal (double-stranded rAAV9) by intraperitoneal injection (I.P.). Tumor microvessel density (MVD) was examined by anti-CD34 staining to evaluate tumor angiogenesis. Compared with the PBS (blank control) group, tumor growth in the high-dose ssrAAV9-Kal group was inhibited by 40% by day 49, and the MVD of tumor tissues was significantly decreased. The results indicate that this therapeutic strategy is a promising approach for clinical cancer therapy and implicate rAAV9-Kal as a candidate for gene therapy of lung cancer.

ATM Inhibition Sensitizes Tumors to High-Dose Irradiation.

Mechanistic studies of high-dose irradiation are important to improve our understanding on how the efficacy of stereotactically delivered high-dose irradiation can be enhanced by therapeutics such as ataxia-telangiesctasia-mutated (ATM) inhibitors. In this issue of Cancer Research, Torok and colleagues found that a single 15 Gy radiation dose eliminated lung tumor growth in mice when ATM was deleted in cancer cells versus when deleted in endothelial cells. These data support the establishment of clinical trials testing ATM inhibitors in combination with highly conformal radiotherapy or high-dose rate brachytherapy.See related article by Torok et al., p. 773.

Intratracheal Administration of siRNA Dry Powder Targeting Vascular Endothelial Growth Factor Inhibits Lung Tumor Growth in Mice

Inhalation therapy using small-interfering RNA (siRNA) is a potentially effective therapeutic strategy for lung cancer because of its high gene-silencing effects and sequence specificity. Previous studies reported that intratracheal administration of siRNA using pressurized metered dose inhalers or nebulizers could suppress tumor growth in murine lung metastatic models. Although dry powder inhalers are promising devices due to their low cost, good portability, and preservability, the anti-tumor effects of siRNA dry powder have not been elucidated. To evaluate the gene-silencing and anti-tumor effects of intratracheally delivered siRNA dry powder, vascular endothelial growth factor-specific siRNA (VEGF-siRNA) dry powder was administered intratracheally to mice with metastatic lung tumors consisting of B16F10 melanoma cells or Lewis lung carcinoma cells. A single intratracheal administration of VEGF-siRNA dry powder reduced VEGF levels in both bronchoalveolar lavage fluid and lung tumor tissue. Furthermore, repeated intratracheal administration of VEGF-siRNA dry powder suppressed the number of visible metastatic foci on the lung surface and tumor area in lung tissues. Taken together, intratracheal administration of siRNA dry powder could be a novel therapeutic strategy for lung cancer through the suppression of specific genes expressed in lung tumor tissue.

Abstract 602: The α7 nicotinic acetylcholine receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy without enhancing the proliferation of lung cancer cells or interfering with paclitaxel-induced antitumor activity

Abstract While cancer chemotherapy continues to significantly contribute to the number of cancer survivors, exposure to these drugs can often result in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms such as numbness, burning, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a microtubule poison that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor for chemotherapy or delay treatment. Our laboratories have previously shown that nicotine, the prototypical nicotinic acetylcholine receptor (nAChR) agonist, can prevent and reverse paclitaxel-induced peripheral neuropathy without increasing lung tumor growth in mice. Our current studies have demonstrated that the α7 nAChR silent agonist R-47 may be a promising preventative and therapeutic treatment for CIPN as well. R-47 (10 mg/kg, p.o.) was administered to male C57BL/6J mice treated with paclitaxel (8 mg/kg, i.p., every other day for a total of four injections). Von Frey filament testing revealed that R-47 can both prevent and reverse paclitaxel-induced mechanical allodynia; the latter effect can be inhibited by the α7 nAChR antagonist methyllycaconitine. In support of these findings, R-47 also prevents the reduction of intra-epidermal nerve fibers in the hind paws of paclitaxel-treated mice. In addition, α7 nAChR knockout mice exhibit a greater and sustained increase in mechanical allodynia after paclitaxel administration when compared to wild-type mice, suggesting the involvement of this receptor subtype in hindering CIPN development and/or maintenance. With regard to cancer, R-47 fails to increase A549 and H460 non-small cell lung cancer cell viability, colony formation, or proliferation alone, and does not interfere with paclitaxel-induced growth arrest, apoptosis, or DNA fragmentation. Most importantly, R-47 does not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These data suggest that the use of an α7 nAChR silent agonist may be a safe and efficacious approach for the prevention and treatment of CIPN. Citation Format: S Lauren Kyte, Wisam Toma, Ganeshsingh Thakur, M Imad Damaj, David A. Gewirtz. The α7 nicotinic acetylcholine receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy without enhancing the proliferation of lung cancer cells or interfering with paclitaxel-induced antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 602.

Blocking fatty acid synthesis reduces lung tumor growth in mice.

Tumors often overexpress enzymes that synthesize fatty acids, but the requirement for fatty acid synthesis in tumor growth is unclear. A new fatty acid–synthesis inhibitor blunts lung tumor growth in mice, which implicates this process as a targetable liability.

High Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives for Onco-Pharmacology.

ObjectivesWe have developed a relevant preclinical model associated with a specific imaging protocol dedicated to onco-pharmacology studies in mice.Materials and MethodsWe optimized both the animal model and an ultrasound imaging procedure to follow up longitudinally the lung tumor growth in mice. Moreover we proposed to measure by photoacoustic imaging the intratumoral hypoxia, which is a crucial parameter responsible for resistance to therapies. Finally, we compared ultrasound data to x-ray micro computed tomography and volumetric measurements to validate the relevance of this approach on the NCI-H460 human orthotopic lung tumor.ResultsThis study demonstrates the ability of ultrasound imaging to detect and monitor the in vivo orthotopic lung tumor growth by high resolution ultrasound imaging. This approach enabled us to characterize key biological parameters such as oxygenation, perfusion status and vascularization of tumors.ConclusionSuch an experimental approach has never been reported previously and it would provide a nonradiative tool for assessment of anticancer therapeutic efficacy in mice. Considering the absence of ultrasound propagation through the lung parenchyma, this strategy requires the implantation of tumors strictly located in the superficial posterior part of the lung.

PDE10 inhibitors suppress lung tumor growth in mice.

e22187 Background: Phosphodiesterase 10 has been studied in the brain as a therapeutic target for psychiatric and neurological conditions, but has not been associated with tumorigenesis. Low levels of PDE10 exist in adult peripheral tissues and we found that 68% of lung tumors (n=19) displayed more than 2-fold increase compared with normal lung, and 26% showed greater than a 10-fold difference. High levels of PDE10 mRNA and protein were also found in human NSCLC cells compared with normal airway epithelium. PDE10 knockdown by siRNA suppressed lung tumor cell growth, as did PDE10 selective inhibitors, papaverine, PQ-10 and Pf-2545920. A novel PDE10 inhibitor, MCI-020 was developed from the indene scaffold of the NSAID, sulindac and was found to potently and selectively inhibit the growth of human NSCLC cells. These observations suggest that PDE10 is essential for lung tumor cell growth and represents a novel therapeutic target for lung cancer. We evaluated pharmacokinetics, tissue distribution and antitumo...

Cimetidine suppresses lung tumor growth in mice through proapoptosis of myeloid-derived suppressor cells

Cimetidine, a histamine type-2 receptor antagonist, is known to inhibit the growth of several tumors in human and animals, however the mechanism of action underlying this effect remains largely unknown. Here, in the mice model of 3LL lung tumor, cimetidine showed significant inhibition of tumor growth. However, an in vitro study demonstrated that cimetidine showed no effect on proliferation, survival, migration and invasion of 3LL cells. We found that cimetidine reduced CD11b+Gr-1+ myeloid derived-suppressive cell (MDSC) accumulation in spleen, blood and tumor tissue of tumor-bearing mice. In vitro coculture assay showed that cimetidine reversed MDSC-mediated T-cell suppression, and improved IFN-γ production. Further investigation demonstrated that the NO production and arginase I expression of MDSCs were reduced, and MDSCs prone to apoptosis by cimetidine treatment. However, MDSC differentiation was not affect by cimetidine. Importantly, although histamine H2 receptor was expressed in MDSC surface, histamine could not reverse the proapoptosis of cimetidine. Moreover, famotidine also did not have this capacity. We found that cimetidine could induce Fas and FasL expression in MDSC surface, and sequentially regulate caspase-dependent apoptosis pathway. Thus, these findings revealed a novel mechanism for cimetidine to inhibit tumor via modulation of MDSC apoptosis.

Enhancement of Carboplatin-Mediated Lung Cancer Cell Killing by Simultaneous Disruption of Glutathione and Thioredoxin Metabolism

Cancer cells (relative to normal cells) show increased steady-state levels of hydroperoxides that are compensated by increased glucose and hydroperoxide metabolism. The current study determined whether inhibitors of glucose and hydroperoxide metabolism could induce chemoradiosensitization by enhancing oxidative stress in lung cancer cells. A549 and NCI-H292 human lung carcinoma cells were treated with 2-deoxy-d-glucose (2DG) combined with carboplatin + ionizing radiation (IR). Lung cancer cells were further sensitized with inhibitors of glutathione (GSH)- and thioredoxin (Trx)-dependent metabolism [buthionine sulfoximine (BSO) and auranofin, respectively] in vitro and in vivo. When 2DG was combined with carboplatin + IR, clonogenic cell killing was enhanced in A549 and NCI-H292 cells, and this combination was more effective than paclitaxel + carboplatin + IR. The thiol antioxidant (N-acetylcysteine, NAC) was capable of protecting cancer cells from 2DG + carboplatin -induced cell killing. Simultaneous treatment of cancer cells with BSO and auranofin, at doses that were not toxic as single agents, also enhanced lung cancer cell killing and sensitivity to 2DG + carboplatin. This treatment combination also increased oxidation of both GSH and Trx, which were inhibited by NAC. Mice treated with auranofin + BSO showed no alterations in circulating leukocytes or red blood cells. Xenograft lung tumor growth in mice was more effectively inhibited by treatment with auranofin + BSO + carboplatin than animals treated with carboplatin or auranofin + BSO alone. These results show in vitro and in vivo that simultaneous inhibition of GSH and Trx metabolism can effectively inhibit lung cancer cell growth and induce chemosensitization by a mechanism that involves thiol-mediated oxidative stress.

Adenovirus-mediated interferon-β gene therapy combined with radiotherapy synergistically inhibits lung tumor growth in mice

7340 Background: The outcome of non-resectable patients with advanced stages of lung cancer (LC) is not satisfying, despite improved chemotherapy and radiotherapy regimes. In other tumors immune-modulatory therapies using various interferons (IFN) delivered via viral vector, have shown some promise. We thus evaluated the use of a replication-deficient adenoviral (Ad) vector encoding for the murine IFN-β gene (Ad.muIFN-β) in mouse LC models in combination with radiotherapy. Methods: L1C2 cells (a mouse LC cell line syngeneic in immune competent BALB/c) were injected either subcutaneously (s.c.) or intraperitoneally (i.p.). Established tumors were treated with a single dose (109 pfu) of Ad.muIFN-β, a control Ad vector (Ad.HSVtk), or saline. Large s.c. flank tumors were additionally treated with radiotherapy (10Gy) at one timepoint. Results: 80% of animals with i.p. and s.c. tumors were cured after a single dose of Ad.IFN-β. However, in animals with large tumor burden (>5% body weight) this strategy was less successful. In large flank tumors, only the combination therapy of a single dose of Ad.muIFN-β and radiotherapy significantly reduced tumor size: With combination therapy, 60% of animals showed complete tumor eradication, whereas monotherapy with Ad.muIFN-β and radiation achieved an eradication rate of 0% and 20%, resp. Conclusions: Local delivery of the murine interferon-β gene via an adenoviral vector provided effective treatment for lung cancers established at various sites, as well as protective acquired immunity. Ad.muIFN-β gene therapy and radiotherapy acted synergistically in lung tumors in mice. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Fresenius Biotech

Adenovirus-mediated interferon-β gene therapy combined with radiotherapy synergistically inhibits lung tumor growth in mice

7340 Background: The outcome of non-resectable patients with advanced stages of lung cancer (LC) is not satisfying, despite improved chemotherapy and radiotherapy regimes. In other tumors immune-modulatory therapies using various interferons (IFN) delivered via viral vector, have shown some promise. We thus evaluated the use of a replication-deficient adenoviral (Ad) vector encoding for the murine IFN-β gene (Ad.muIFN-β) in mouse LC models in combination with radiotherapy. Methods: L1C2 cells (a mouse LC cell line syngeneic in immune competent BALB/c) were injected either subcutaneously (s.c.) or intraperitoneally (i.p.). Established tumors were treated with a single dose (109 pfu) of Ad.muIFN-β, a control Ad vector (Ad.HSVtk), or saline. Large s.c. flank tumors were additionally treated with radiotherapy (10Gy) at one timepoint. Results: 80% of animals with i.p. and s.c. tumors were cured after a single dose of Ad.IFN-β. However, in animals with large tumor burden (>5% body weight) this strategy was less successful. In large flank tumors, only the combination therapy of a single dose of Ad.muIFN-β and radiotherapy significantly reduced tumor size: With combination therapy, 60% of animals showed complete tumor eradication, whereas monotherapy with Ad.muIFN-β and radiation achieved an eradication rate of 0% and 20%, resp. Conclusions: Local delivery of the murine interferon-β gene via an adenoviral vector provided effective treatment for lung cancers established at various sites, as well as protective acquired immunity. Ad.muIFN-β gene therapy and radiotherapy acted synergistically in lung tumors in mice. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Fresenius Biotech

Correlation of immune response with clinical stage in Lewis lung tumor-bearing mice.

The effect of Lewis lung tumor growth in mice on the induction of primary immune response to SRBC, was investigated by PFC assay for measuring antibody activity and by footpad test as a correlate for delayed type hypersensitivity reactions. With the appearance of micrometastases in the lungs there was a decline in the humoral and cellular immune response to the SRBC. An increase of number and size of metastases in the lungs led to a further depression of the immune reactivity. Since the reduction of general immune response in mice bearing this tumor is not due to a direct influence of tumor cells, it might be assumed that suppressor cells or factors, are actively abrogating the general and also the tumor directed immune reactions.