Hyaluronic acid as a macromolecular crowding agent for production of cell-derived matrices.

Cell-derived matrices (CDMs) provide an exogenous source of human extracellular matrix (ECM), with applications as cell delivery vehicles, substrate coatings for cell attachment and differentiation, and as biomaterial scaffolds. However, commercial application of CDMs has been hindered due to the prolonged culture time required for sufficient ECM accumulation. One approach to increasing matrix deposition in vitro is macromolecular crowding (MMC), which is a biophysical phenomenon that limits the diffusion of ECM precursor proteins, resulting in increased ECM accumulation at the cell layer. Hyaluronic acid (HA), a natural MMC highly expressed in vivo during fetal development, has been shown to play a role in ECM production, but has not been investigated as a macromolecule for increasing cell-mediated ECM deposition in vitro. In the current study, we hypothesized that HA can act as a MMC, and increase cell-mediated ECM production. Human dermal fibroblasts were cultured for 3, 7, or 14 days with 0%, 0.05%, or 0.5% high molecular weight HA. Ficoll 70/400 was used as a positive control. SDS-PAGE, Sircol, and hydroxyproline assays indicated that 0.05% HA-treated cultures had significantly higher mean collagen deposition at 14 days, whereas Ficoll 70/400-treated cultures had significantly lower collagen production compared to the HA and untreated controls. However, fluorescent immunostaining of ECM proteins and quantification of mean gray values did not indicate statistically significant differences in ECM production in HA or Ficoll 70/400-treated cultures compared to untreated controls. Raman imaging (a marker-free spectral imaging method) indicated that HA increased ECM deposition in human dermal fibroblasts. These results are consistent with decreases in CDM stiffness observed in Ficoll 70/400-treated cultures by atomic force microscopy. Overall, these results indicate that there are macromolecule- and cell type- dependent effects on matrix assembly, turnover, and stiffness in cell-derived matrices. STATEMENT OF SIGNIFICANCE: Cell-derived matrices (CDMs) are versatile biomaterials with many regenerative medicine applications, including as cell and drug delivery vehicles and scaffolds for wound healing and tissue regeneration. While CDMs have several advantages, their commercialization has been limited due to the prolonged culture time required to achieve CDM synthesis in vitro. In this study, we explored the use of hyaluronic acid (HA) as a macromolecular crowder in human fibroblast cell cultures to support production of CDM biomaterials. Successful application of macromolecular crowding will allow development of human cell-derived, xeno-free biomaterials that re-capitulate the native human tissue microenvironment.

A microfluidic trap array for longitudinal monitoring and multi-modal phenotypic analysis of individual stem cell aggregates.

Three-dimensional pluripotent stem cell (PSC) cultures have the ability to undergo differentiation, self-organization, and morphogenesis to yield complex, in vitro tissue models that recapitulate key elements of native tissues. These tissue models offer a system for studying mechanisms of tissue development, investigating disease mechanisms, and performing drug screening. It remains challenging, however, to standardize PSC aggregate differentiation and morphogenesis methods due to heterogeneity stemming from biological and environmental sources. It is also difficult to monitor and assess large numbers of individual samples longitudinally throughout culture using typical batch-based culture methods. To address these challenges, we have developed a microfluidic platform for culture, longitudinal monitoring, and phenotypic analysis of individual stem cell aggregates. This platform uses a hydrodynamic loading principle to capture pre-formed stem cell aggregates in independent traps. We demonstrated that multi-day culture of aggregates in this platform reduces heterogeneity in phenotypic parameters such as size and morphology. Additionally, we showed that culture and analysis steps can be performed sequentially in the same platform, enabling correlation of multiple modes of analysis for individual samples. We anticipate this platform being applied to improve abilities for phenotypic analysis of PSC aggregate tissues and to facilitate research in standardizing culture systems in order to dually increase the yield and reduce the heterogeneity of PSC-derived tissues.

The role of adhesion junctions in the biomechanical behaviour and osteogenic differentiation of 3D mesenchymal stem cell spheroids.

Osteogenesis of mesenchymal stem cells (MSC) can be regulated by the mechanical environment. MSCs grown in 3D spheroids (mesenspheres) have preserved multi-lineage potential, improved differentiation efficiency, and exhibit enhanced osteogenic gene expression and matrix composition in comparison to MSCs grown in 2D culture. Within 3D mesenspheres, mechanical cues are primarily in the form of cell-cell contraction, mediated by adhesion junctions, and as such adhesion junctions are likely to play an important role in the osteogenic differentiation of mesenspheres. However the precise role of N- and OB-cadherin on the biomechanical behaviour of mesenspheres remains unknown. Here we have mechanically tested mesenspheres cultured in suspension using parallel plate compression to assess the influence of N-cadherin and OB-cadherin adhesion junctions on the viscoelastic properties of the mesenspheres during osteogenesis. Our results demonstrate that N-cadherin and OB-cadherin have different effects on mesensphere viscoelastic behaviour and osteogenesis. When OB-cadherin was silenced, the viscosity, initial and long term Young's moduli and actin stress fibre formation of the mesenspheres increased in comparison to N-cadherin silenced mesenspheres and mesenspheres treated with a scrambled siRNA (Scram) at day 2. Additionally, the increased viscoelastic material properties correlate with evidence of calcification at an earlier time point (day 7) of OB-cadherin silenced mesenspheres but not Scram. Interestingly, both N-cadherin and OB-cadherin silenced mesenspheres had higher BSP2 expression than Scram at day 14. Taken together, these results indicate that N-cadherin and OB-cadherin both influence mesensphere biomechanics and osteogenesis, but play different roles.

Biomaterial strategies for controlling stem cell fate via morphogen sequestration.

Protein sequestration plays an essential role in maintaining stem cell populations in the native stem cell niche. Both pluripotent and adult stem cells require the sustained presentation of numerous bioactive growth factors and other soluble cues to potentiate cell fate decisions and morphogenic events. Consequently, methods of natural protein sequestration employed by the stem cell niche present attractive strategies for developing novel protein delivery vehicles and engineering biomimetic stem cell microenvironments that enhance morphogen bioactivity. In this review, we will explore the role of protein sequestration in the native stem cell niche and how it has inspired the design of several classes of materials that exploit natural protein sequestration to effectively maintain stem cell populations and direct stem cell fate. We will also highlight several recent developments in protein sequestering biomaterials, in which material strategies to sequester complex mixtures of endogenously secreted proteins are also being investigated.

Phenotypic analysis of familial breast cancer: comparison of BRCAx tumors with BRCA1-, BRCA2-carriers and non-familial breast cancer.

AIMS: Women with inherited pathogenic mutations in the BRCA1 or BRCA2 genes have up to an 85% risk of developing breast cancer in their lifetime. However, only about 20% of familial breast cancer is attributed to mutations in BRCA1 and BRCA2, while a further 5-10% are attributed to mutations in other rare susceptibility genes such as TP53, STK11, PTEN, ATM and CHEK2. Despite extensive efforts to explain the missing heritability of this disease, the majority of familial clustering in breast cancer remains largely unexplained. We aim to analyze the pathology of familial cases of which no pathogenic mutation is yet identified. METHODS: We compared the pathological phenotype of BRCA1/BRCA2 negative familial breast cancer (BRCAx) to BRCA1-positive, BRCA2-positive and sporadic cases without a family history. Age-adjusted analysis is summarized in odd's ratios and confidence intervals for tumor type, grade, lymph node, ER and HER2 status. RESULTS: We found non-familial cases to be more likely to be ER positive (P = 0.041) as compared with BRCAx tumors. More cases of lobular carcinoma were found with BRCAx as compared to BRCA1 tumors (P = 0.05). After multivariate logistic regression analysis, BRCAx tumors are more likely ER positive (P = 0.001) and HER2 positive (P = 0.047) in comparison to BRCA1. Conversely, BRCAx cases are less likely to be ER positive (P = 0.02) but more likely to be HER2 positive (P = 0.021) as compared with BRCA2 tumors. CONCLUSION: Our findings suggest that BRCA1, BRCA2 and BRCAx tumors differ in phenotype from non-familial and familial BRCA1-positive and BRCA2-positive tumors. Further studies will need to be performed in this important population in order to develop strategies for early detection and prevention.

Dysregulated STAT1-SOCS1 control of JAK2 promotes mammary luminal progenitor cell survival and drives ERα(+) tumorigenesis.

We previously reported that STAT1 expression is frequently abrogated in human estrogen receptor-α-positive (ERα(+)) breast cancers and mice lacking STAT1 spontaneously develop ERα(+) mammary tumors. However, the precise mechanism by which STAT1 suppresses mammary gland tumorigenesis has not been fully elucidated. Here we show that STAT1-deficient mammary epithelial cells (MECs) display persistent prolactin receptor (PrlR) signaling, resulting in activation of JAK2, STAT3 and STAT5A/5B, expansion of CD61(+) luminal progenitor cells and development of ERα(+) mammary tumors. A failure to upregulate SOCS1, a STAT1-induced inhibitor of JAK2, leads to unopposed oncogenic PrlR signaling in STAT1(-/-) MECs. Prophylactic use of a pharmacological JAK2 inhibitor restrains the proportion of luminal progenitors and prevents disease induction. Systemic inhibition of activated JAK2 induces tumor cell death and produces therapeutic regression of pre-existing endocrine-sensitive and refractory mammary tumors. Thus, STAT1 suppresses tumor formation in mammary glands by preventing the natural developmental function of a growth factor signaling pathway from becoming pro-oncogenic. In addition, targeted inhibition of JAK2 may have significant therapeutic potential in controlling ERα(+) breast cancer in humans.

Familial breast cancer genetic testing in the West of Ireland.

AIMS: The majority of hereditary breast and ovarian cancers are associated with highly penetrant mutations in two genes: BRCA 1 and 2. Our aim was to investigate the prevalence and types of BRCA mutations in patients from the West of Ireland. METHODS: A retrospective cohort study was undertaken that included all patients from the counties, Mayo, Sligo, Galway, Roscommon, and Clare, who were referred to the National Centre for Medical Genetics (NCMG) for testing for mutations in BRCA 1 or 2 between 2000 and 2010. Data including age, symptoms, family history, Manchester score, and test results were recorded and analysed using SPSS. RESULTS: The NCMG received 380 referrals from the Western seaboard, including 148 for diagnostic testing and 232 for predictive evaluation. Sixty-five patients did not attend for assessment. Two hundred and fifty-six patients fulfilled criteria for genetic counselling, which was accepted by 184, of whom 127 proceeded to testing. Predictive tests were more often declined than diagnostic [41 (46 %) vs. 16 (17 %)]. Ten mutations in BRCA 1 were identified in 20 patients (15 families), including Exon 1-23del (3 families); Exon 14-20del (2 families) and E143X (2 families). Six mutations in BRCA 2 were identified in 15 patients (12 families) including 8525delC (n = 2 families) and 8205-1G>C (n = 3 families). Patients with positive results had significantly higher Manchester scores than those with negative tests [median 25.5 (12-48) vs. 20 (8-37), p = 0.042, Mann-Whitney U test]. CONCLUSION: To identify patients with highly penetrant variants, referrals should be made with strict adherence to guidelines. Counselling should be individualised to counteract intrinsic psychological barriers to testing.

Characterization of BMS-911543, a functionally selective small-molecule inhibitor of JAK2.

We report the characterization of BMS-911543, a potent and selective small-molecule inhibitor of the Janus kinase (JAK) family member, JAK2. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in cell lines dependent upon JAK2 signaling, and had little activity in cell types dependent upon other pathways, such as JAK1 and JAK3. BMS-911543 also displayed anti-proliferative responses in colony growth assays using primary progenitor cells isolated from patients with JAK2(V617F)-positive myeloproliferative neoplasms (MPNs). Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2 signaling, with sustained pathway suppression being observed after a single oral dose. At low dose levels active in JAK2-dependent PD models, no effects were observed in an in vivo model of immunosuppression monitoring antigen-induced IgG and IgM production. Expression profiling of JAK2(V617F)-expressing cells treated with diverse JAK2 inhibitors revealed a shared set of transcriptional changes underlying pharmacological effects of JAK2 inhibition, including many STAT1-regulated genes and STAT1 itself. Collectively, our results highlight BMS-911543 as a functionally selective JAK2 inhibitor and support the therapeutic rationale for its further characterization in patients with MPN or in other disorders characterized by constitutively active JAK2 signaling.

Cascade screening in BRCA1/2 mutation carriers.

Genetic counselling for families with BRCA1 & 2 has been available in Ireland since 1998. We describe the follow-on cascade from the initial 29 index cases that tested positive for either gene. 28 of the index cases were female and 1 was male. Their combined sibship and offspring totalled 125 and 129 respectively. Of the 125 siblings, 21/72 (29%) females and 10/53 (19%) males came forward for counselling and all were tested. Of the 129 at risk offspring, 56 (43%) [25 females and 31 males] were over 18 years and therefore were eligible for testing. 20/25 (80%) females and 6/31 (19%) males came for counselling and all bar one female was tested. In summary, 31/125 (25%) at risk siblings were tested and 25/56 (45%) offspring were tested. Only one person, a daughter of an affected individual who attended clinic, declined testing. The remaining 197 (77%) individuals have not come forward for counselling. Our results suggest that a) there is a low-moderate testing rate in Ireland when compared to other European centres; and that b) daughters of BRCA1/2 carriers are more likely to come forward for testing than female siblings. Specific factors which appeared to influence attendance included poor dissemination of information among families about the test; and lower levels of communication among siblings than within the nuclear family.

Predictive testing for BRCA1 and 2 mutations: a male contribution.

BACKGROUND: Management strategies for women carrying BRCA1 and 2 mutations are becoming clearer and predictive testing for a known family mutation is commonly undertaken. Implications for men are not as clear and they participate less frequently. PATIENTS AND METHODS: Twenty-six men from 10 extended families underwent predictive testing. Their motivation, reaction and outcome were studied. Subjects had appropriate pre- and post-test counselling. Informed consent was obtained before predictive testing for known deleterious mutations. DNA analysis followed standard procedures. RESULTS: Eighteen tested positive and eight negative. Four had adverse psychological reactions and three reneged on their commitments to impart results. The spouse of another man had an adverse psychological reaction to the disclosure of his positive result. Two, already suffering from prostate cancer, were phenocopies and paternal lineage transmission was unexpectedly determined in another. Risk was removed from 33 offspring and confirmed for 56. CONCLUSIONS: Complex themes associated with genetic testing are confirmed and the spectrum extended. Men appear to understand the importance of participating in this process. Methods of avoiding adverse reactions merit further study along with other aspects of the process.

Quantitative interrogation of micropatterned biomolecules by surface force microscopy.

Synthetic biomaterials are widely used in medical implants with success in improving and extending quality of life. However, these materials were not originally designed to interact with cells through specific signaling pathways. As a result, the interaction with the body is mediated through passive adsorption of a disorganized protein monolayer. Next generation biomaterials have been proposed to be active in modifying the biological response of the host through the incorporation of specific biorecognition moieties. An important tool in the development of these novel active biomaterials is the scanning force microscope (SFM). The SFM allows for interrogation of bioactive biomaterials in mapping or spectroscopic modes. In this work, micropatterned protein surfaces were prepared using biomolecules implicated in wound healing. The surfaces were imaged via SFM and the specific binding forces between surface associated biomolecules and antibody functionalized tips were quantified.

Constrained cell recognition peptides engineered into streptavidin.

Streptavidin is widely used as an adaptor molecule in diagnostics, separations, and laboratory assay applications. We have here engineered cell adhesive peptides into the three-dimensional scaffolding of streptavidin to convert streptavidin into a functional protein. The mutations did not alter refolding or tetramer assembly and the slow biotin dissociation rate of wild-type streptavidin was retained. The peptide targets were hexapeptide sequences derived from osteopontin and fibronectin that contain the RGD cell adhesion sequence. Cell binding assays directly demonstrated that rat aortic endothelial cells and human melanoma cells adhered to surfaces coated with either of the two RGD streptavidin mutants in a dose-dependent fashion. Wild-type streptavidin displayed no significant cell binding activity. Inhibition studies with soluble RGD peptides confirmed that the cell adhesion was RGD mediated. Further inhibition studies with antibodies directed against alphavbeta3 demonstrated that the RGD-streptavidin interaction was primarily mediated by this integrin with melanoma cells. These results demonstrate that peptide recognition sequences can be engineered into accessible surface regions of streptavidin without disrupting biotin binding properties. This approach to introducing secondary functional activities into streptavidin may improve streptavidin's utility in existing applications or provide new technology opportunities.

Merocyanine 540 solubilized as an ion pair with cationic surfactant in nonpolar solvents: spectral and photochemical properties.

Merocyanine 540 (MC) is an anionic dye that is used to photopurge the bone marrow of leukemia cells. Under these conditions it is localized mostly in cell membranes, which may affect its photochemical reactivity. We investigated the photochemistry of MC dissolved as a hydrophobic ion pair with a hexadecyltrioctadecylammonium cation in cyclohexane, trimethylpentane and toluene as well as in propylene carbonate, CH3CN, C2H5OH and D2O. In organic solvents, the absorption and fluorescence spectra of MC were strongly red-shifted compared with aqueous solutions. The fluorescence was also more intense despite aggregation that occurred in some solvents. Aggregation strongly affects the spectral and photochemical properties of MC, especially in aliphatic hydrocarbons in which distinctive H-type aggregates are formed. Hydrophobic MC is a moderate photosensitizer of singlet molecular oxygen (1O2). The following values for 1O2 quantum yields were calculated based on 1O2 phosphorescence relative to 1O2 generation by Rose Bengal: approximately 0.12 in trimethylpenthane, approximately 0.13 in cyclohexane, 0.045 in EtOH, 0.039 in toluene, 0.007 in CH3CN and approximately 3 x 10(-4) in D2O. The H-aggregates of MC in cyclohexane and trimethylpentane are better 1O2 producers than monomeric MC. The above 1O2 quantum yields are corrected for self-quenching because MC is an efficient 1O2 quencher (17 x 10(7) M-1 s-1 in CH3CN, 6.8 x 10(7) M-1 s-1 in D2O, 5.2 x 10(7) M-1 s-1 in EtOH, and 1.4 x 10(7) M-1 s-1 in toluene). Merocyanine undergoes photodegradation, a solvent-dependent process that proceeds faster when the dye is aggregated. The initial photodegradation rate is much slower in organic solvents than in water, but photodegradation products accumulated during longer irradiation may increase the rate in most solvents. Higher photostability and better photosensitization by MC in hydrophobic nonpolar solvents suggest that the killing of leukemia cells via a photodynamic mechanism may operate mostly in cell membranes. In contrast, any cytotoxic products from photodecomposition may be important in hydrophilic cell compartments. Our data show the spectral and photochemical properties of MC in a pure hydrophobic environment.

Smart and biofunctional streptavidin.

The high affinity recognition of biotin and biotinylated molecules has made streptavidin one of the most important components in diagnostics and laboratory kits. While it is extremely useful as the native protein, there are many applications where its function can be improved re-engineering the subunits. We review here our efforts to construct streptavidin tetramers that have 'smart' recognition capabilities, and which display functional peptide sequences. These smart and biofunctional streptavidin derivatives can 'talk' to cells, and 'listen' to external signals which control capture and release of biotinylated molecules.

Purification and characterization of a tumor lipid-mobilizing factor.

Cancer patients with weight loss showed urinary excretion of a lipid-mobilizing factor (LMF), determined by the ability to stimulate lipolysis in isolated murine epididymal adipocytes. Such bioactivity was not detectable in the urine of cancer patients without weight loss or in normal subjects. The LMF was purified using a combination of ion exchange, exclusion, and hydrophobic interaction chromatographies to give a single component of apparent Mr 43,000, which showed homology in amino acid sequence with human plasma Zn-alpha2-glycoprotein. Both substances showed the same mobility on denaturing and nondenaturing gels and the same chymotrypsin digestion pattern, both stained heavily for carbohydrate, and they showed similar immunoreactivity. Polyclonal antisera to human plasma Zn-alpha2-glycoprotein was also capable of neutralization of the bioactivity of human LMF in vitro. Using competitive PCR to quantify expression of Zn-alpha2-glycoprotein, we found that only those tumors that were capable of producing a decrease in carcass lipid expressed mRNA for Zn-alpha2-glycoprotein. These results provide strong evidence to suggest that tumor production of Zn-alpha2-glycoprotein is responsible for the lipid catabolism seen in cancer patients.

Catabolic factors in cancer cachexia.

A lipid mobilizing factor has been purified from a cachexia-inducing mouse colon adenocarcinoma (MAC16) using a combination of ion exchange (Mono Q), exclusion (Superose) and reverse phase hydrophobic chromatography. The purification process led to a 3,500-fold increase in the specific activity. Serum from mice bearing the MAC16 tumour contained antibodies reactive with fractions containing lipid mobilizing activity and detectable as a 24 kDa immunoreactive band on Western blotting. Serum from mice transplanted with a related tumour, MAC13, not producing cachexia, did not contain antibodies. A similar immunoreactive band was detectable in the urine of patients with cancer cachexia, but was absent from the urine of normal subjects. A monoclonal antibody produced by fusion of splenocytes from mice bearing the MAC16 tumour with mouse Balb/c myeloma cells attenuated the development of cachexia in mice transplanted with the MAC16 tumour and inhibited tumour growth. These results suggest that the M(r) 24 kDa antigen may be important in tumour growth and cachexia.

Induction of muscle protein degradation and weight loss by a tumor product.

Splenocytes from mice bearing a cachexia-inducing tumor (MAC16) have been fused with mouse myeloma cells to produce hybridomas, which have been cloned to produce antibody reactive to a material which copurified with a lipid-mobilizing factor isolated from the same tumor. The monoclonal antibody has been used to investigate factors potentially involved in the development of cachexia. The major protein detectable by immunoprecipitation of a partially purified lipid-mobilizing factor was M(r) 69,000, whereas Western blotting showed two bands of M(r) 69,000 and M(r) 24,000. Although the monoclonal antibody did not neutralize lipid-mobilizing activity in an in vitro assay, it did neutralize a serum factor capable of protein degradation in isolated gastrocnemius muscle. Affinity purification of MAC16 tumor homogenates using the monoclonal antibody yielded two immunoreactive bands of M(r) 69,000 and M(r) 24,000, which were further fractionated on a hydrophobic column (C8). This material was capable of inducing tyrosine release from isolated gastrocnemius muscle, and the effect could be blocked with the monoclonal antibody. The two immunoreactive bands from the hydrophobic column were capable of inducing weight loss in mice, whereas nonimmunoreactive fractions had no effect on body weight. The M(r) 24,000 species had a unique amino acid sequence, whereas the M(r) 69,000 species gave the same sequence as the M(r) 24,000 material, together with that for albumin. The M(r) 24,000 species contained carbohydrate, and lectin blotting showed a strong reaction with wheat germ and Erythrina crystagalli agglutinins. This suggests that the material is a glycoprotein or proteoglycan that shows strong binding affinity for albumin, possibly through the carbohydrate residues.

Characterization of a cancer cachectic factor.

Cancer cachexia is a syndrome of progressive wasting which has been suggested to be mediated by tumour-necrosis factor-alpha, interleukins 1 and 6, interferon-gamma and leukaemia-inhibitory factor. It has proved difficult to correlate levels of tumour-necrosis factor-alpha and interleukin-6 with cancer cachexia, and the weight loss induced by leukaemia-inhibitory factor may be due to toxicity. In the murine adenocarcinoma MAC16, cachexia is mediated by circulatory catabolic factors, which we have now isolated using an antibody cloned from splenocytes of mice transplanted with the MAC16 tumour, with a delayed cachexia. The material is a proteoglycan of relative molecular mass 24K which produces cachexia in vivo by inducing catabolism of skeletal muscle. The 24K material was also present in urine of cachectic cancer patients, but was absent from normal subjects, patients with weight loss due to trauma, and cancer patients with little or no weight loss. This suggests that cachexia in mice and humans may be produced by the same material.

Purification and characterization of a lipid-mobilizing factor associated with cachexia-inducing tumors in mice and humans.

A scheme is described for the purification of a lipid-mobilizing factor from a cachexia-inducing murine tumor (MAC16) using a combination of ion exchange (Mono Q), exclusion (Superose), and hydrophobic (C8) chromatography. This process yields an active material with an apparent molecular weight of 24,000 with an overall purification of 3,500 from the tumor homogenate and representing 0.005% of the total protein present. The material tends to aggregate to high molecular mass, is acidic (pI < 4), and displays heterogeneity of charge as evidenced by a broad elution profile on ion exchange and exclusion chromatography and multiple peaks on hydrophobic columns. The purified material was heat and alkali (pH 10.4) labile and activity could be completely inhibited by sulfatase, suggesting that the negative charge could arise from sulfate residues. There was no evidence that the material possessed triglyceride lipase activity. Animals transplanted with the MAC16 tumor and with a delayed weight loss contained in their serum antibodies that recognized a M(r) 24,000 band on Western blots. This material copurified with the lipid-mobilizing factor. Such antibodies were not present in the serum of mice transplanted with the MAC13 tumor, which does not induce cachexia, suggesting that the antibodies were directed to the induction of cachexia rather than the tumor itself. Urine from patients with cancer cachexia also contained a lipid-mobilizing factor which adhered to DEAE-cellulose and gave an apparent M(r) of 24,000 by exclusion chromatography. Western blotting using serum from MAC16 tumor-bearing animals showed the presence of a band of M(r) 24,000 in such fractions, which was not detected using serum from mice bearing the MAC13 tumor. This band was not present in Western blots of urine from normal subjects. The fact that serum from mice bearing the MAC16 tumor can detect the human lipid-mobilizing activity suggests a high degree of structural similarity between the two and raises the possibility that cachexia in humans may be caused by the same species as in the mouse.