Spermine dialdehyde, a novel ex vivo purging agent for both allogenic and autologous bone marrow transplantations

The use of ex vivo purging agents has shown to be beneficial for both allogeneic and autologous bone marrow transplantations. We have shown previously that spermine dialdehyde (SDA), an oxidized product of spermine, when used in phosphate-buffered saline (PBS) preferentially inhibits T-cell proliferation while sparing myeloid cells. Lethally irradiated mice were rescued by reconstitution with SDA-treated allogeneic marrow and showed no sign of graft-vs-host disease (GVHD). In this paper, we show by HPLC analysis that SDA degraded rapidly in PBS but remained intact in saline. Administered in saline, SDA was more inhibitory on leukemic cell lines than normal myeloid or T-cells. Lethally irradiated mice receiving a syngeneic bone marrow leukemic cell mixture treated ex vivo with SDA in saline did not manifest leukemia. Thus, the preferential inhibitory effect of SDA and its degraded products in different buffers suggest that SDA could act as a novel purging agent for both allogeneic and autologous bone marrow transplantations.     

Characterization of monoclonal antibodies directed against the alpha-subunit of the human IgE high-affinity receptor

Effective ex vivo purging techniques can decrease the likelihood of infusing bone marrow contaminated with leukemic cells during autologous transplantation. In preliminary studies, OL(1)p53, a 20-mer phosphorothioate oligonucleotide directed against p53 mRNA, decreased the number of acute myelogenous leukemia (AML) cells in vitro, suggesting a possible role for OL(1)p53 in purging bone marrow harvests of leukemia cells. To demonstrate that OL(1)p53 was nontoxic to hematopoietic progenitor cells, normal bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h, and hematopoietic progenitor cell survival was determined by in vitro colony assays. OL(1)p53 had no toxic effect on the growth of either myeloid (CFU-GM) or erythroid (BFU-E) progenitor cells. OL(1)p53 was then used to ex vivo purge bone marrow harvests from nine patients with either AML or myelodysplastic syndrome (MDS). Bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h before transplantation. The median times posttransplantation for the patient to recover an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet transfusion independence were 30 days and 56 days, respectively. Incubation of bone marrow cells with OL(1)p53 had no detrimental effect on the growth of hematopoietic progenitor cells, and transplantation of autologous bone marrow cells treated with the phosphorothioate oligonucleotide, OL(1)p53, resulted in successful recovery of circulating neutrophils following high-dose therapy in patients with AML or MDS. The data show that OL(1)p53 can be used safely to purge autologous bone marrow harvests from patients with leukemia.     

Leukemic predisposition of mice transplanted with gene-modified hematopoietic precursors expressing flt3 ligand

flt3/flk-2 ligand (FL) is a cytokine that exhibits synergistic activities in combination with other early acting factors on subpopulations of hematopoietic stem/progenitor cells. In addition to normal hematopoietic precursors, expression of the FL receptor, flt3R, has been frequently demonstrated on the blast cells from patients with acute B-lineage lymphoblastic, myeloid, and biphenotypic (also known as hybrid or mixed) leukemias. Because many of these leukemic cell types express FL, the possibility has been raised that altered regulation of FL-mediated signaling might contribute to malignant transformation or expansion of the leukemic clone. In humans, FL is predominantly synthesized as a transmembrane protein that must undergo proteolytic cleavage to generate a soluble form. To investigate the consequences of constitutively expressing the analogous murine FL isoform in murine hematopoietic stem/progenitor cells, lethally irradiated syngeneic mice (18 total) were engrafted with post-5-fluorouracil-treated bone marrow cells transduced ex vivo with a recombinant retroviral vector (MSCV-FL) encoding murine transmembrane FL. Compared with control mice (8 total), MSCV-FL mice presented with a mild macrocytic anemia but were otherwise healthy for more than 5 months posttransplant (until 22 weeks). Subsequently, all primary MSCV-FL recipients observed for up to 1 year plus 83% (20 of 24) of secondary MSCV-FL animals that had received bone marrow from asymptomatic primary hosts reconstituted for 4 to 5 months developed transplantable hematologic malignancies (with mean latency periods of 30 and 23 weeks, respectively). Phenotypic and molecular analyses indicated that the tumor cells expressed flt3R and displayed B-cell and/or myeloid markers. These data, establishing that dysregulated expression of FL in primitive hematopoietic cells predisposes flt3R+ precursors to leukemic transformation, underscore a potential role of this cytokine/receptor combination in certain human leukemias.     

Reduced efficacy of allogeneic versus syngeneic fibroblasts modified to secrete cytokines as a tumor vaccine adjuvant

We examined the relative efficacy of allogeneic versus syngeneic fibroblasts admixed with tumor cells as a vaccine to induce antitumor T-cell reactivity. Allogeneic (3T3) or syngeneic (BLK) fibroblasts transfected to secrete equivalent amounts of GM-CSF were admixed with either D5 melanoma or MCA 207 sarcoma and inoculated s.c. into the flanks of C57BL/6 mice. Vaccine-primed lymph node (LN) cells were examined for in vivo antitumor reactivity in an adoptive transfer model. At fibroblast: tumor cell ratios of < or=1, allogeneic and syngeneic granulocyte macrophage colony-stimulating factor-secreting fibroblasts enhanced T-cell reactivity to tumor cells. However, at ratios of 2.4, the adjuvant effect induced by granulocyte macrophage colony-stimulating factor was not evident. Instead, we observed increased alloreactivity of primed LN cells against 3T3 targets as assessed by cytotoxicity and cytokine release assays, which was not observed with syngeneic fibroblasts. Moreover, with increasing numbers of allogeneic fibroblasts, there was a skewing of the T-cell Vbeta repertoire. These latter cells responded to tumor stimulation with the release of greater amounts of interleukin 10, which may account for the diminished antitumor reactivity observed in vivo. Allogeneic fibroblasts transduced to secrete interleukin 2 or IFN-gamma also induced diminished tumor reactivity of primed LN cells. Syngeneic fibroblasts are superior to allogeneic fibroblasts as vehicles to deliver cytokines in tumor vaccines.     

Induction of pre-B lymphoid leukemia following reconstitution of lethally irradiated mice with v-erb-B virus-infected bone marrow progenitor cells

We have previously shown that v-erb-B contained within a recombinant murine retroviral vector is capable of transforming pre-B lymphocytes (M. Miller, A. K. Kennewell, and G. Symonds, Leukemia, 6: 18-28, 1992) and early erythroid precursor cells [blast-forming units (erythroid) (M. Miller, A. Kennewell, Y. Takayama, A. Bruskin, J. M. Bishop, G. Johnson, and G. Symonds, Oncogene, 5: 1125-1131, 1990)] in vitro. To determine the sites and nature of v-erb-B-induced transformation in vivo, the hematopoietic systems of lethally irradiated mice were repopulated with v-erb-B-infected bone marrow. All mice became moribund within 4-12 weeks of reconstitution, with a median onset of disease at 6 weeks. Histopathological and flow cytometric evaluation of tissues from diseased mice, as well as morphological and phenotypic analysis (cytochemical as well as molecular) of the cell lines established from the mice, revealed that all but one of the mice examined at postmortem had developed a pre-B lymphoid leukemia or lymphoma. Abnormally high levels of mast cells in the spleen and bone marrow of the remaining mouse indicated a mast cell disease. The development of pre-B lymphoid malignancy in the majority of the reconstituted mice indicates a marked predisposition of v-erb-B to transform cells of the pre-B lymphoid lineage. The reconstitution of lethally irradiated mice with v-erb-B virus-infected bone marrow provides a model system for the analysis of events involved in the initiation and maintenance of acute lymphoid leukemia.     

Activation of gp130 signaling in vivo by the IL-6 super-agonist K-7/D-6 accelerates repopulation of lymphoid organs after irradiation

Stimulation of the gp130 signaling pathway by IL-6 is known to contribute significantly to hematopoietic expansion in vitro, mostly in combination with other cytokines. In the present study we have investigated whether a similar effect can be observed also in vivo using short-term assays in which irradiated mice were analyzed for repopulation of lymphoid organs. Mice were injected with a combination of soluble IL-6Ralpha either with wild-type (wt) human IL-6 or with an IL-6 variant, called K-7/D-6, that shows a 70-fold higher IL-6Ralpha affinity. We observed that while wt IL-6 was able to induce a partial effect only in combination with IL-3, K-7/D-6 bypassed the need for IL-3 and yielded complete recovery. In lethally irradiated mice reconstituted with syngeneic bone marrow cells K-7/D-6 strongly accelerated the repopulation of thymus and spleen and hastened blood neutrophil recovery. These results underscore the potential of the gp130 signaling pathway in hematopoietic reconstitution after myeloablative regimens and open the possibility to fully exploit it with a super-active IL-6 variant.     

Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL-11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.     

Aberrant dipeptidyl peptidase IV (DPP IV/CD26) expression in human hepatocellular carcinoma

The HOX11/TCL3 homeobox gene was identified at the breakpoint region in pediatric T-cell acute lymphoblastic leukemia harboring 10q24 chromosomal translocations. We previously reported that primary murine bone marrow cells transduced ex vivo with a recombinant HOX11-containing retrovirus, MSCV-HOX11, gave rise to cell lines at high frequency having characteristics of early myeloid cells. Cell lines were also established from the bone marrow and spleen of transplant recipients sacrificed 5 months after engraftment with MSCV-HOX11-transduced bone marrow cells. These latter lines, which exhibited a more differentiated myelomonocytic phenotype, harbored proviruses encoding a smaller HOX11 protein. None of the mice that received HOX11-expressing bone marrow cells or myeloid cell lines developed leukemia during 6-month observation periods. Here, we report that two bone marrow transplant recipients eventually developed T-cell acute lymphoblastic leukemia-like malignancies at 7 and 12 months posttransplant, indicating that progression to a fully malignant state required additional mutations. One tumor synthesized full-length HOX11 whereas the other expressed the smaller version of the protein. The smaller HOX11 protein suffered a carboxyl-terminal truncation. We subsequently constructed MSCV-based retroviral vectors expressing deleted forms of HOX11 and identified an amino-terminal region that was dispensible for generation of myeloid cell lines having a similar phenotype as those induced by full-length HOX11. We thus conclude that regions near the amino and carboxyl termini of HOX11 are not essential for transforming function, nor do they appear to determine the lineage or stage of differentiation of the target cell for transformation.     

Granulocyte colony-stimulating factor reduces the capacity of blood mononuclear cells to induce graft-versus-host disease: impact on blood progenitor cell transplantation

The feasibility of transplanting peripheral blood mononuclear cells (PBMC) from granulocyte colony-stimulating factor (G-CSF)-treated normal human donors to myeloablated allogeneic hosts has been demonstrated recently. The current work examined the ability of recombinant G-CSF to alter peripheral blood T-cell function and graft-versus-host disease (GVHD) in a murine model of allogeneic G-CSF-mobilized PBMC transplantation. Administration of recombinant G-CSF to C57BL/Ka mice markedly increased the capacity of PBMC to reconstitute lethally irradiated syngeneic hosts. T- and B-lineage lymphocytes were depleted about 10-fold in the bone marrow of the treated mice, and the T-cell yield in the blood was increased about fourfold. The ability of PBMC or purified CD4+ and CD8+ T cells to induce acute lethal GVHD in irradiated BALB/c mice was reduced after the administration of G-CSF. This was associated with decreased secretion of interferon gamma and interleukin-2 (IL-2) and an increased secretion of IL-4. The donor cell inoculum, which was most successful in the rescue of irradiated allogeneic hosts, was the low-density fraction of PBMC from G-CSF-treated mice. These low-density cells were enriched for CD4-CD8-NK1.1+ T cells and secreted about 10-fold more IL-4 than the unfractionated cells from the G-CSF-treated donors.     

Hypoxia induces high-mobility-group protein I(Y) and transcription of the cyclooxygenase-2 gene in human vascular endothelium

BACKGROUND: Animals reconstituted with allogeneic whole bone marrow (WBM) are often tolerant of donor-specific solid organ grafts. Clinical application of bone marrow transplantation in solid organ transplantation has been limited, however, principally by graft-versus-host disease. We previously demonstrated that hematopoietic stem cells (HSCs) reconstitute lethally irradiated allogeneic mice without producing graft-versus-host disease. The purpose of this study was to determine whether tolerance to solid organ grafts could be induced in mice reconstituted with HSCs. METHODS: BALB/c mice were lethally irradiated and reconstituted with allogeneic C57BL/Ka, Thy-1.1 WBM or HSCs. An isolated group was given a limited number of HSCs (250 cells) and a subpopulation of allogeneic cells known to facilitate HSC engraftment (facilitators). C57BL/Ka, Thy-1.1 neonatal heart grafts were placed in reconstituted animals either at the time of hematopoietic transplant or 35 days later. Third-party C3H grafts were placed over 2 months after hematopoietic reconstitution. Tolerance was defined as the persistence of cardiac contraction for the duration of evaluation (125-270 days). RESULTS: All surviving mice that were reconstituted with C57BL/Ka, Thy-1.1 HSCs, WBM, or HSCs and facilitators were tolerant of C57BL/Ka grafts long-term. Third-party C3H grafts placed in reconstituted animals were rejected by day 12, whereas those placed in unmanipulated mice were rejected by day 9. CONCLUSION: These data indicate that tolerance to concurrently or subsequently placed solid organ grafts can be reliably achieved with limited numbers of purified HSCs in a model where immunocompetence to third-party major histocompatibility complex antigens is delayed but intact.     

Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell-dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell-stimulating agents, such as poly I:C or recombinant mouse interferon-gamma. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.     

Long-term expression, systemic delivery, and macrophage uptake of recombinant human glucocerebrosidase in mice transplanted with genetically modified primary myoblasts

A critical requirement for treatment of Gaucher disease via systemic delivery of recombinant GC is that secreted enzyme be in a form available for specific takeup by macrophages in vivo. In this article we investigated if transplanted primary myoblasts can sustain expression of human GC in vivo and if the secreted transgene product is taken up by macrophages. Transduced primary murine myoblasts were implanted into syngeneic C3H/HeJ mice. The results demonstrated that transplanted mice sustained long-term expression of transferred human GC gene in vivo. Furthermore, human GC is secreted into the circulation of mice transplanted with syngeneic primary myoblasts retrovirally transduced with human GC cDNA. The transplanted primary myoblasts differentiate and fuse with adjacent mature myofibers, and express the transgene product for up to 300 days. Human GC in the circulation reaches levels of 20-280 units/ml of plasma. Immunohistochemical studies of the target organs revealed that the secreted human GC is taken up by macrophages in liver and bone marrow. Immunochemical identification of reisolated myoblasts from transplanted mice showed that MFG-GC-transduced cells also survived as muscle stem cells in the implanted muscle. These results present in encouraging prospect for the treatment of Gaucher disease.     

Significant contribution of spleen cells in mediating the lethal effects of endotoxin in vivo

Two closely related, histocompatible mouse strains that have marked differences in both in vitro and in vivo responses to endotoxin were used to evaluate the contribution of lymphoid cells to the lethal effect of endotoxin. C3H/HeJ mice are endotoxin resistant, whereas C3H/HeN mice are endotoxin sensitive. In vitro spleen cell mitogenic responses to endotoxin were similar in untreated mice and in mice that received sublethal irradiation (450 R) followed by reconstitution with autologous spleen cells. Reconstitution with spleen cells from the related strain produced chimeric animals with spleen cell mitogenic activity like that of the donor strain. When chimeric animals were subjected to a lethal challenge of endotoxin, their response was markedly altered by the transferred lymphoid cells. C3H/HeJ animals reconstituted with C3H/HeN cells became more endotoxin sensitive, whereas C3H/HeJ cells became more endotoxin resistant. These results indicate that spleen cells play a significant, detrimental role in endotoxin-induced lethality.     

A stable RAG1-RAG2-DNA complex that is active in V(D)J cleavage

A well-known characteristic of gamma delta T cells is that they are produced in waves during ontogeny, with cells expressing T-cell receptor V gamma 5 appearing early in fetal thymic ontogeny, followed by V gamma 6, then by other gamma delta T-cell types. In addition, evidence exists to suggest that the potential of haemopoietic precursors to generate different types of gamma delta T cells changes in ontogeny. We have used these observations as the basis for an extensive study of the potential for haemopoietic precursors isolated from fetal liver, neonatal spleen and adult bone marrow to reconstitute severe combined immunodeficient (SCID) mice. Mice that were reconstituted as newborns with fetal liver cells most closely resembled normal C.B-17 mice with respect to both lymphocyte numbers and subsets, while mice reconstituted with adult bone marrow had fewer cells than normal mice. This deficit spanned both T and B cells in all organs examined. Among the gamma delta T-cell subsets examined, the ability to reconstitute V gamma 4+ cells was particularly dependent on the ontogenic age of the reconstituting presursors, with fetal liver cells having the greatest capacity to generate V gamma 4+ cells, and adult bone marrow cells the least. The vast majority of the T cells produced in the reconstituted mice were of donor origin, and the level of reconstitution was found to be dependent upon some factor other than the presursor frequency.     

Antiviral cytotoxic activity across a species barrier in mixed xenogeneic chimeras: functional restriction to host MHC

Reconstitution of lethally irradiated mice with a mixture of mouse and rat bone marrow cells (mouse + rat-->mouse) results in mixed xenogeneic chimerism and donor-specific tolerance. The current study demonstrates that mouse and rat T lymphocytes that have developed in xenogeneic chimeras are restricted to Ag presentation by mouse, but not rat, APC. Restriction to host Ags results in functional immunocompetence with generation of antiviral cytotoxic activity in vivo, within and across species barriers. These data demonstrate for the first time that the host thymus is sufficient to support development and positive selection of functional cross-species T lymphocytes. The superior immunocompetence, as compared with fully xenogeneic (rat-->mouse) chimeras, may prove to be of significant benefit in the clinical application of xenotransplantation to solid organ transplantation and immune reconstitution for AIDS.     

Eradication of pre-established lymphoma using herpes simplex virus amplicon vectors

Herpes simplex virus amplicon vectors expressing RANTES (HSVrantes) and the T-cell costimulatory ligand B7.1 (HSVB7.1) were studied for their ability to elicit a tumor-specific T-cell response in a murine lymphoma model. HSVB7.1- and HSVrantes-transduced EL4 cells expressed high levels of B7.1 and RANTES as analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. Inoculation of ex vivo HSVB7.1 transduced cells in syngeneic mice resulted in regression of both transduced cells and nontransduced cells inoculated contralaterally. Direct intratumoral injection of HSVB7.1 and/or HSVrantes alone or in combination into established EL4 tumors led to complete tumor regression in injected tumors as well as in nontransduced contralaterally implanted tumor, whereas control tumors or tumors injected with HSVlac expressing beta-galactosidase did not regress. Maximal protection was achieved with combined injection of HSVB7.1 and HSVrantes; mice showing tumor regression were resistant to rechallenge with parental EL4 cells, and tumor cell-specific cytolytic T-cell activity was observed in mice demonstrating regression. HSV amplicon-mediated delivery of immune effector molecules may represent a useful strategy for immunotherapy in the setting of pre-existing tumor.     

Enhanced acceptance of regenerating bone marrow of random bred newborn mice by random bred adult mice

Regenerating bone marrow of newborn random bred Sabra mice (9-13 days old) was obtained by the administration of two consecutive i.p. injections of hydroxyurea (HU) (2 x 100 mg/kg body wt), three days prior to collection of the marrow cells. The bone marrow of HU-treated newborn mice was assayed for CFU-S, CFU-C and plasma-clot-diffusion-chamber (PCDC) progenitor cells. A fourfold content of CFU-S was found in the regenerating bone marrow compared with that of the control marrow, while the level of CFU-C and PCDC progenitor cells was the same in treated and untreated newborn mice. In lethally irradiated adult, random bred Sabra recipient mice, transfused with regenerating bone marrow from newborn mice, the initial survival rate was greater than in irradiated animals receiving normal newborn marrow (75% as against 50%); marrow repopulation, 10-14 days after transfusion, was also greater in the former than in the latter group of animals (1.5-2x10(6) nucleated cells per femur as compared with 08.-2x10(5)). The bone marrow of these groups of mice was assayed for CFU-S, CFU-C and PCDC progenitor cells; with a cell inoculum of 5 x 10(4) i.v., 10(5) in vitro and 5 x 10(4) per DC, respectively, pluripotent and committed stem cells were detected in the experimental group and were lacking in control recipients. Regenerating bone marrow of newborn mice was also transfused into lethally irradiated splenectomized recipients. In this experimental group there was high mortality, low marrow repopulation and lack of CFU-S (5-10 x 10(4) cell inoculum). The results of this study indicate that, despite genetic differences among random bred Sabra mice, regenerating bone marrow of newborn mice "takes better" than normal marrow in lethally irradiated recipients. Improved marrow acceptance is possibly due to the increased content of activated CFU-S and/or pre-CFU-S in the regenerating bone marrow     

Misrepresentation of publications by applicants for radiology fellowships: is it a problem?

In light of the recently described experimental technique of in vivo bone reconstitution with biotechnologic methods (from bone marrow stromal cells) and the prefabrication flap procedures, the possibility to obtain autologous bone growth in a myocutaneous flap, thus creating a composite osteomyocutaneous preformed flap, is postulated. Human bone marrow stromal cells were delivered into the latissimus dorsi of athymic mice by a porous hydroxyapatite ceramic model. Eight weeks after the implantation, histologic examination revealed the presence of spongious bone tissue. A simple myocutaneous flap was thus transformed into a composite osteomyocutaneous flap. This flap is called the biotechnologic prefabricated flap, because it was the result of ex vivo expanded osteogenic precursor cells and in vivo bone tissue neoformation. The shape of the bone flap was exactly the same as the shape of the ceramic model used. A possible clinical application may be the correction of skeletal defects. The advantages of this procedure are simple surgical execution, the possibility of preshaping the graft to the exact characteristics of the defect, and the availability of autogenous donor tissue without donor site morbidity.     

Removal of carcinoma cells from contaminated bone marrow using the lipophilic cation rhodamine 123

Autologous bone marrow transplants for solid tumor treatment are severely limited by the potential presence of residual cancer cells in the reinfused bone marrow and can lead to future tumor recurrence. This article presents a novel method of removing carcinoma cells from bone marrow with contaminating cancer cells. This method is based on our previous studies demonstrating that carcinoma cells have a higher uptake of lipophilic cations such as rhodamine 123 than their normal epithelial counterparts. When the relative differences in rhodamine 123 uptake are quantified, carcinoma cell lines demonstrated a 7.4-21 times greater uptake of rhodamine 123 than normal mouse bone marrow cells. More important, when normal bone marrow cells and carcinoma cell lines are mixed to simulate carcinoma-contaminated bone marrow, individual cell populations continue to exhibit characteristic and identifiable relative differences (10-20 times) in rhodamine 123 uptake. Differential sorting of bone marrow/carcinoma cell mixtures with respect to rhodamine 123 fluorescence intensity resulted in the removal of 95-99% of the "contaminating carcinoma cells." The recovered bone marrow cells were fully viable as ascertained by their ability to form splenic colonies. In our preliminary experiments, sorted bone marrow cells transplanted into lethally irradiated C57BL6 mice allowed the mice to survive for more than 8 months. In light of these promising results, we propose that lipophilic cations may play a role in the purification of autologous bone marrow used in transplants for patients with advanced solid tumors.