Phase III trial of nonpegylated liposomal doxorubicin in combination with trastuzumab and paclitaxel in HER2-positive metastatic breast cancer.

BACKGROUND: Nonpegylated liposomal doxorubicin liposomal doxorubicin, (Myocet™; Sopherion Therapeutics, Inc Canada, and Cephalon, Europe) (NPLD; Myocet(®)) in combination with trastuzumabHerceptin(®) (Hoffmann-La Roche) has shown promising activity and cardiac safety. We conducted a randomized phase III trial of first-line NPLD plus trastuzumab and paclitaxel (Pharmachemie B.V.) (MTP) versus trastuzumab plus paclitaxel (TP) in patients with human epidermal growth factor 2 receptor (HER2)-positive metastatic breast cancer. PATIENTS AND METHODS: Patients were randomly assigned to NPLD (M, 50 mg/m(2) every 3 weeks for six cycles), trastuzumab (T, 4 mg/kg loading dose followed by 2 mg/kg weekly), and paclitaxel (P, 80 mg/m(2) weekly) or T + P at the same doses until progression or toxicity. The primary efficacy outcome was progression-free survival (PFS). RESULTS: One hundred and eighty-one patients were allocated to receive MTP, and 183 to TP. Median PFS was 16.1 and 14.5 months with MTP and TP, respectively [hazard ratio (HR) 0.84; two-sided P = 0.174]. In patients with estrogen receptor (ER)- and progesterone receptor (PR)-negative tumors, PFS was 20.7 and 14.0 months, respectively [HR 0.68; 95% confidence interval (CI) 0.47-0.99]. Median overall survival (OS) was 33.6 and 28.9 months with MTP and TP, respectively (HR 0.79; two-sided P = 0.083). In ER- and PR-negative tumors, OS was 38.2 and 27.9 months, respectively (HR 0.63; 95% CI 0.42-0.93). The frequency of adverse events was higher with MTP, but there was no significant difference in cardiac toxicity between treatment arms. CONCLUSION(S): The trial failed to demonstrate a significant clinical improvement with the addition of M to TP regimen. The clinical benefit observed in an exploratory analysis in the ER- and PR-negative population deserves consideration for further clinical trials. CLINICAL TRIAL NUMBER: NCT00294996.

Differential locomotion of long- and short-term IL-2-activated murine natural killer cells in a model matrix environment.

Tumour infiltration by activated natural killer (A-NK) cells is a pre-requisite for tumour eradication by adoptive NK cell transfer. Extravasated A-NK cells do not always succeed in reaching the crucial target cell conjugation. Therefore, we wished to study A-NK cell locomotion and interactions with melanoma cells in a matrix environment (Matrigel) by electron, confocal and fluorescence microscopy. Two distinct patterns of A-NK cell-mediated matrix disintegration were revealed during incubation of tumour cells and A-NK cells in Matrigel: (1) A-NK cells pre-cultured for 5 days altered the homogeneous texture of the Matrigel, an initial microporous appearance became a loose filamentous meshwork by 24 h. Matrix degrading protease inhibitors could not fully prevent this, but could delay the process; and (2) A-NK cells pre-cultured for 6 days or more, instead formed large excavations in the Matrigel leaving the remaining matrix less affected compared to the effects by the younger A-NK cells. By histochemical staining with Cupromeronic Blue, the excavations were shown to contain proteoglycan material. Protease inhibitors had no discernable effect on the development of the excavations. The conspicuous capacity of A-NK cells to disintegrate extracellular matrix and the formation of large excavations seems only partially to depend on matrix-degrading proteases. Formation of extracellular proteoglycan material is suggested to facilitate A-NK cell locomotion within a matrix environment.

uPA and uPAR contribute to NK cell invasion through the extracellular matrix.

BACKGROUND: The urokinase plasminogen activator (uPA) system has been implicated in cellular invasiveness of tumor cells and immune cells. Herein we provide evidence for the production by natural killer (NK) cells of both uPA and its receptor (uPAR). MATERIALS AND METHODS: Western blot analysis, RTPCR, casein/plasminogen zymography, and fluorescence microscopy were employed to detect uPA and uPAR on NK cells. NK cell invasiveness was examined using Matrigel invasion assays. RESULTS: NK cell uPA appeared at its characteristic molecular weights, is enzymatically active in casein/plasminogen zymography, and is recognized by monoclonal antibodies. uPAR was detected by RTPCR and fluorescence microscopy. Matrigel invasion assays demonstrated an active role of uPA in NK cell invasion. CONCLUSION: The uPA system contributes to extracellular matrix (ECM) degradation by NK cells, which may be essential for NK cell accumulation into metastases, and may be prerequisite for their killing of tumor cells following NK cell adoptive transfer.

Expression of neutrophil collagenase (MMP-8) in Jurkat T leukemia cells and its role in invasion.

BACKGROUND: Previous studies have shown that MMP-8, the neutrophil collagenase, was expressed in neutrophils, chondrocytes and rheumatoid synovial fibroblasts. MATERIALS AND METHODS: We used semi-quantitative RT-PCR analysis, Western blotting, and immunofluorescence assays to determine the expression of MMP-8 in Jurkat T cells. RESULTS: We have determined the expression of MMP-8 from Jurkat cells and the down-regulation of its expression by genistein, a principal soy isoflavone. Genistein inhibited the invasion of Jurkat cells through a model basement membrane by about 75%, similar to the inhibition by BB-94, a synthetic MMP inhibitor. Genistein also down-regulated the expression of MMP-13, but slightly up-regulated the expression of TIMP-1 and TIMP-2. CONCLUSIONS: Our findings documented for the first time the expression of the neutrophil collagenase by a T-cell line. We also determined the inhibition of Jurkat cell invasion by genistein, which was in part mediated through the regulation of the expression of MMPs and TIMPs.

Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer.

In this article we report about the role that tumor structure and extracellular matrix (ECM) may play in immunotherapy and in gene therapy using adenoviruses. We performed studies in a rat model for colorectal cancer, CC531, and in specimens of human colorectal cancer. The tumors were composed of two compartments, tumor cell nests surrounded by stromal cells. ECM proteins were expressed in the stromal part, where the blood vessels were also located. Furthermore, in several tumors, the tumor cell nests were surrounded by basal membrane-like structures. Therefore, in vascular approaches to treat cancer, therapeutic agents on their route to tumor cells may be hampered by ECM to reach tumor cells. We found that immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor. Adenoviruses, when intravascularly injected, did not reach tumor cells in the CC531 rat model. Tumor cells were only infected, and even then in limited numbers, in cases of intratumoral injection. We hypothesize that ECM in a tumor is a barrier both for immune cells and for adenoviruses to make direct contact with these tumor cells, and thus limits colorectal tumor therapy.

Expression of matrix metalloproteinases and their inhibitors by rat NK cells: inhibition of their expression by genistein.

In this study, we describe rat NK cell-derived MMPs including membrane-type MMPs (MT-MMPs) and tissue inhibitors of MMP (TIMPs). RT-PCR analysis from cDNA of rat A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-7, MMP-10, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. The RNK-16 cells expressed mRNA for MMP-7, MMP-10, MMP-11, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2, in addition to MMP-3 and MMP-13. Western blot analysis confirmed proteins for MT1-MMP and MT2-MMP in RNK-16 cells. TIMP-1 in rat A-NK cells was present at molecular mass of 34-kDa protein which may represent a highly glycosylated form. Genistein, a natural isoflavone found in soybeans, inhibited proliferation of RNK-16 cells in dosage dependent manner. In addition, it down-regulated the expression of MMP-13, MT1-MMP, TIMP-1 and TIMP-2. Moreover, genistein greatly impaired the ability of RNK-16 cells to invade through a model basement membrane. This effect might be mediated by the observed down-regulation of MMP-13 and MT1-MMP.

Cooperation of urokinase plasminogen activator and matrix metalloproteinases in NK cell invasion.

We have previously investigated the role of the urokinase plasminogen activator (uPA) system in NK cell invasion. We have also studied NK cell-derived matrix metalloproteinases (MMPs) in extracellular matrix (ECM) degradation. We now report that both enzyme systems cooperate in NK cell invasion. Zymographic analyses detected uPA in RNK-16 cell conditioned media (CM) with the same molecular weights as the uPA we have previously shown to be associated with the rat NK cell urokinase plasminogen activator receptor. The combination of aprotinin, an inhibitor of plasmin, and Batimastat (BB94), an inhibitor of MMPs, in Matrigel invasion assays showed a more potent inhibitory effect on NK cell invasion than either inhibitor alone. Finally, a down regulation of uPA mRNA was noted following RNK-16 stimulation with collagen IV, fibronectin, and laminin.

Tumor blood supply and tumor localization by adoptively transferred IL-2 activated natural killer cells.

The circulatory pattern of IL-2 activated natural killer (A-NK) cells was studied in C57BL/6 mice bearing 10 day-old pulmonary and subcutaneous (s.c.) metastases of the B16 melanoma in order to evaluate the roles of the concentration of A-NK cells in the blood and of tumor blood flow on accumulation of A-NK cells in tumors. Kinetic studies of the presence of A-NK cells in peripheral blood after adoptive transfer revealed that these cells rapidly disappear from the blood. Via intravital microscopy of animals with exposed lung tissue, we have shown that the vast majority of transferred A-NK cells become efficiently arrested within the lung microcirculation at their first encounter with this organ, thereby explaining the fast disappearance of the cells from the bloodstream. Despite the low number of A-NK cells circulating in the blood, systemically injected A-NK cells (20 million per mouse) localized significantly (70-80 million cells/g) into most pulmonary metastases within 8-16 hours. In contrast, very few A-NK cells (< 0.2 million cells/g) were found in the s.c. metastases. Based on measurements of tumor blood flow (showing a classic inverse relationship between tumor size and tumor blood flow) and the blood concentration of A-NK cells, we estimated the highest intratumoral density of A-NK cells that theoretically can be generated by A-NK cells transported to the tumor by way of the blood. In s.c. tumors, the observed density of A-NK cells was at all times lower (10-50 fold) than the estimated density, indicating that only a few percent of the A-NK cells arriving at these tumors become retained in them. In contrast, the observed density of A-NK cells in pulmonary metastases was at all times higher (2-3 fold) than the estimated density. This finding indicates that A-NK cells might not reach the pulmonary metastases solely by way of the blood stream. In conclusion, i.v. injected A-NK cells become immediately entrapped in the lungs and, consequently, circulate poorly. While lung metastases become significantly infiltrated by i.v. injected A-NK cells, metastases in organs down-stream from the lungs become poorly infiltrated. We hypothesize that only a part of the A-NK cells found in lung metastases 8-16 hours following injection reach these metastases by way of the blood-vascular system. They might also migrate into the metastases from the surrounding normal lung tissue.

Regulation of IFN-gamma production following 2B4 activation in human NK cells.

IFN-gamma is a cytokine that regulates various functions of the immune system. The major producers of IFN-gamma are T cells and NK cells. 2B4 is a novel activating receptor expressed on all human NK cells, a subset of CD8+ T cells, monocytes and basophils. Activation of human NK cells through surface 2B4 enhances NK cell cytolytic function and secretion of IFN-gamma. We have examined the regulation of IFN-gamma production by the human NK cell line YT upon activation through surface 2B4. Our data indicate that ligation of surface 2B4 by mAb C1.7, that specifically recognizes 2B4, induces transcriptional activation of IFN-gamma. Partial inhibition of transcription did not prevent the transcriptional upregulation of IFN-gamma. S1 nuclease protection analysis indicated that transcriptional activation as well as mRNA stability may account for the increased production of IFN-gamma by human NK cells following 2B4 stimulation.

2B4 stimulation of YT cells induces natural killer cell cytolytic function and invasiveness.

2B4 is a surface molecule found on all human natural killer (NK) cells, a subset of CD8+ T cells, monocytes and basophils. It was originally identified on mouse NK cells and the subset of T cells that mediate non-major histocompatibility complex (MHC)-restricted killing. Recently,9 we have cloned the human homologue of 2B4 (h2B4) and found h2B4 to also mediate non-MHC-restricted cytotoxicity. In this study, we examine h2B4 in regulating various functions of NK cells using a human NK cell line YT, with monoclonal antibody (mAb) C1.7, an antibody that specifically recognizes h2B4. Ligation of surface 2B4 with mAb C1.7 increases YT's ability to destroy tumour cells. In the presence of mAb C1.7, the production of interferon-gamma (IFN-gamma) by YT cells is greatly enhanced. Engagement of surface 2B4 by mAb C1.7 downregulates the expression of h2B4 at the cell surface as well as the expression of h2B4 mRNA. Also, signalling through h2B4 causes the increased expression of matrix metalloproteinase-2, a member of the matrix degrading proteinase family. Thus, in addition to modulating cytolytic function and cytokine production of NK cells, activation through surface 2B4 may play a role in upregulating the machinery for degradation of extracellular matrices to promote invasion of the tumour by NK cells.

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo.

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA-uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136-143). This 8-mer capped peptide (A6) inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of A6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from A6-treated animals. A6 treatment also resulted in a decrease in factor VIII-positive tumor microvessel hot-spots. These results identify a new epitope in uPA that is involved in the uPA-uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.

Blood level of B and CD4+ lymphocytes measured before induction of an experimental tumor in rats predicts tumor progression and survival.

After an initial series of experiments indicated that early responses of B lymphocytes were important in controlling tumor metastases in two rat models of cancer (N. Quan et al., Cancer Res., 59: 1080-1089, 1999), the present study assessed whether differences in the number of B lymphocytes that are normally present in different individual rats before any tumor development could predict tumor growth, metastases, and length of survival when tumor challenge subsequently occurred. Repeated baseline measures of several circulating lymphocyte subtypes (i.e., natural killer, B, CD4+, CD8+ lymphocytes) were made in individual inbred WAG rats before any introduction of tumor cells, and stable baselines for these subtypes were found. Animals were then injected with 2 x 10(6) CC531 tumor cells (a syngeneic tumor) into the leg, and the size of the resulting primary tumor measured. Primary tumors were surgically removed 6-7 weeks after tumor-cell injection, and animals then followed until death from metastases. In two experiments, the size of the primary tumor as well as the length of time that animals survived correlated with the pretumor percentage of certain lymphocyte subtypes in peripheral blood before tumor-cell injection. Baseline percentage of B lymphocytes was significantly negatively correlated with the size of the primary tumor and was positively correlated with the duration of survival. Baseline percentage of CD4+ lymphocytes showed the opposite relationship, being positively correlated with tumor size and negatively correlated with survival time, although these correlations were lower than those for B lymphocytes. Percent B lymphocytes in circulation also declined during tumor development. In summary, a high percentage of endogenous peripheral blood B lymphocytes predicted growth of smaller primary tumors and longer survival after experimental tumor induction in a rat model, further suggesting that B lymphocytes are involved in protection against development of certain tumors.

Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors.

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.

A novel drug delivery system using IL-2 activated NK cells and Zyn-linked doxorubicin.

Adoptively transferred IL-2 activated NK (A-NK) cells selectively accumulate within tumor metastases which recommends them as vehicles for locoregional drug delivery. Zyn-Linkers are membrane-binding lipophilic dyes which can be coupled by a variety of conjugation chemistries to therapeutic agents. We have previously demonstrated that A-NK cells labeled with PKH26 are able to accumulate within established B16 melanoma pulmonary metastases by 16 h at a concentration of over 600 cells/mm2 of tumor tissue (Basse et al. J. Exp. Med. 174: 479 1991). Zyn-205 is a prodrug in which doxorubicin is attached to a similar Zyn-Linker through an acid-sensitive bond. We have optimized the ex vivo labeling conditions and found that a 10 min incubation with 25 microM Zyn-205 results in the uptake of over 10(8) drug molecules per cell with no effect on either cell viability or cytolytic activity up to 24 h after labeling. Given these parameters, the amount of drug which may be carried to and concentrated in metastatic lesions represents a local concentration of approximately 15 microM. In addition, A-NK cells carrying Zyn-Linked doxorubicin at an equivalent dose of 25 micrograms/kg was therapeutically comparable to a systemic dose of 8 mg/kg (320x more) in the 3LL model of experimental metastasis. These data indicate that A-NK cells bearing Zyn-Linked chemotherapeutic agents represent a unique and feasible method to target chemotherapeutic agents to cancer metastases and that therapeutic doses can be attained without unwanted systemic exposure.

Proteasome inhibitor and lymphocyte function: partial inhibition of cell-mediated cytotoxicity and implication that the lymphocyte proteasome may contain multiple chymotryptic domains.

The multicatalytic proteinase complex or proteasome possesses at least 4 distinct proteolytic activities. We have previously reported that the chymotrypsin-like activity of the rat natural killer cell proteasome may play a role in natural killer (NK) cell-mediated cytotoxicity or IL-2 activated NK (A-NK) cell-mediated cytotoxicity. Using a series of novel, Cephalon, Inc, synthetic proteasome inhibitors (CEP-1508, CEP-1612 and CEP-3117) which have been reported to be specific for the chymotrypsin-like activity of the proteasome, we have further investigated the possible role of the proteasome, with emphasis on the chymotryptic activity components, in cell-mediated cytotoxicity. We now report that these compounds can inhibit the rat NK proteasome in a dose dependent manner. Nevertheless, there is only a 50% inhibition of A-NK cell-mediated cytotoxicity. These results confirm and extend our previous results that the proteasome contributes, at least in part, to cell-mediated cytotoxicity. However, as anticipated, since multiple molecular pathways contribute to cell-mediated cytotoxicity, the proteasome contributes only partially to NK cell-mediated cytolytic reactivity. The exact role of the proteasome in NK cell-mediated killing, and whether single or multiple chymotryptic domains function directly or indirectly, remains to be fully determined.

Matrix metalloproteinases of human NK cells.

We have previously reported that MMP-2 and MMP-9 are present in rat A-NK cells, and have recently documented that additional MMPs are present in rodent A-NK cells. To our knowledge only proMMP-9 has previously been reported for human NK and A-NK cells. Herein, we report for the first time the presence of MMP-2 and MT1-MMP in human NK cells. The importance of these enzymes for the migration of A-NK cells into tumor metastases is of great potential relevance. MMPs may be rate limiting in A-NK cells, following their adoptive transfer, to traverse basement membrane and accumulate within established cancer metastases, a likely pre-requisite to their cytolytic function. Human NK cells express and produce MMP-2, MMP-9, MT1-MMP and the inhibitor TIMP-1. Moreover, human A-NK cells degrade the extracellular matrix equivalent (Matrigel) in a seemingly IL-2 dependent manner. It is therefore likely that A-NK cell MMPs play crucial roles in contributing to A-NK cell localisation and positioning the cells in vivo to allow for triggering their cytolytic potential.

Hepatocyte growth factor (HGF) and receptor (c-met) in normal and malignant astrocytic cells.

BACKGROUND: Hepatocyte growth factor (HGF) is a multifunctional peptide that binds to a specific receptor, c-met. Both HGF and c-met have been identified in normal brain and on glial tumors. The purpose of this study is to further define the biologic importance of HGF and c-met on normal and malignant glial cells grown in vitro. MATERIALS AND METHODS: Nine human malignant glioma-derived tumor cell cultures and cultures of astrocytes derived from normal brain were examined for c-met and HGF transcripts using Northern blot or RT-PCR analysis. Cellular invasiveness was quantitated by mechanical assay and mitogenesis was determined by cell count. RESULTS: C-met was expressed in five of seven malignant glioma-derived tumor cell cultures and in both normal astrocyte cultures. HGF transcript was not detected in any of the cell cultures. HGF supplementation enhanced invasiveness in c-met positive cell lines and did not alter cellular mitogenesis in the assayed cultures. CONCLUSIONS: These findings suggest that HGF is a potent stimulator of invasiveness in c-met positive malignant glioma-derived tumor cells and is not an active cytokine with regards to in vitro glial cell proliferation. HGF may therefore stimulate glioma cellular invasion in vivo through binding to its receptor and by activating tyrosine kinase secondary messengers.

Enhanced anti-metastatic efficacy of IL-2 activated NK (A-NK) cells with novel benzothiazoles.

We have previously shown that A-NK cells when locoregionally administered accumulate within established cancer metastases and establish direct contact with both tumor cells and microvascular endothelial cells. Nevertheless, the accumulation of adoptively transferred A-NK cells into established cancer metastases is not sufficient for therapeutic efficacy in the B16 melanoma model. We have therefore attempted to enhance the anti-metastatic therapeutic efficacy of adoptively transferred A-NK cells with standard anticancer chemotherapeutic agents. We have found that chemoimmunotherapy with A-NK cells plus cyclophosphamide to be more effective than A-NK cell adoptive immunotherapy alone. We have now built on these findings, by examining the ability of novel biologic response modifiers (low molecular weight benzothiazole compounds) to augment adoptive immunotherapy with A-NK cells. Two compounds KB-R4107 (4-methoxy-2-(4-t-butylphenyl)benzothiazole) and KB-R4250 (4-methoxy-2-(4-trifluoromethylphenyl)benzothiazole) enhanced reduction of B16 melanoma pulmonary metastases mediated by A-NK cell adoptive immunotherapy. Both compounds were administered for 5 days prior to administration of A-NK cells at 100 mg/kg p.o. All experimental groups initially contained at least 7 animals and were examined for tumor burden on day 10. With B16 melanoma cells administered on day 0 and A-NK cells administered on Day 4, KB-R4107 and KB-R4250 yielded on average a 64% and 52% reduction in metastatic burden, respectively compared to an average 17% reduction using A-NK cells alone. In contrast these compounds did not diminish metastatic burden when administered alone. KB-R4107 and KB-R4250 are therefore low molecular weight, heterocyclic, biological response modifiers which can augment the anti-metastatic therapeutic effect of adoptively transferred A-NK cells.

Evidence for involvement of B lymphocytes in the surveillance of lung metastasis in the rat.

These studies examined the composition of lymphocytes within the lung after the introduction of tumor cells that metastasize to the lung in rats. i.v. delivery of MADB106 tumor cells into syngeneic Fischer 344 rats caused dose- and time-dependent development of lung tumors, with surface metastases evident 7 days after injection and markedly increased 11 days after injection. The total number of lymphocytes recovered from the lung was increased 11 days after injection but not 7 days after injection. When lymphocytes from the lung, spleen, and blood were subjected to fluorescence-activated cell sorting analysis, the most conspicuous change was an increase in the percentage of CD45RA+ cells (i.e., B lymphocytes in the rat) in the lung, with no changes seen in the percentage of natural killer (NKR-P1+), CD4+, or CD8+ cells in the lung. Analysis of the time course showed that B lymphocytes increased in the lung soon after i.v. tumor injection, with an initial peak seen 6 h after injection. Rapid influx of B lymphocytes into lung after i.v. tumor cell injection was also observed in another syngeneic tumor model, i.e., after injection of CC531 cells into WAG rats. To determine whether the influx of B lymphocytes into the lung might participate in tumor surveillance, a high dose of antibody (100 microg) to rat B lymphocytes was given to immunoneutralize these cells; this produced an increase in lung tumors in both models. Finally, Fischer 344 rats were given a s.c. injection of MADB106 tumor cells that made them resistant to lung tumors when given a later i.v. injection of these tumor cells. These animals were found to have an elevated level of B lymphocytes residing in the lung associated with the resistance to lung tumor. These findings suggest that early responses of B lymphocytes are important in protection against tumor development in two rat models of cancer.