Abstract The embryolethal effects of phosphonacetyl‐L‐aspartic acid (PALA) are markedly gestational stage‐specific in the Swiss albino mouse. Embryos are most sensitive to the lethal effects of PALA on days 7 and 8 of gestation, with embryonic LD 50 's of 9 and 8 mg/kg, respectively. In contrast, the embryonic LD 50 on day 10 of gestation is 144 mg/kg. Following an IP dose of (acetyl‐ 14 C)‐PALA to pregnant mice, approximately threefold higher levels of radioactivity were present in day 8 than in day 10 embryonic tissue, whereas the radioactive content of placentas from day 10 pregnant animals was significantly higher than in placentas from day 8 pregnant mice. Similarly, L‐aspartate transcarbamylase (ATCase) activity was greater in maternal spleen, placentas, and embryos on day 8 than on day 10 of gestation, and PALA treatment produced a greater inhibition of ATCase in embryonic tissue on day 8 than on day 10; however, inhibition of placental ATCase activity was more pronounced on day 10 than on day 8. Neither single nor multiple doses of uridine (UR) given orally to pregnant mice on day 8 of gestation were effective in reducing day 8 PALA embryolethality. Carbamyl‐L‐aspartic acid, given in the drinking water of pregnant mice on days 7–9 of gestation, reduced the day 8 embryolethality of PALA from 100% to approximately 50%. In a similar experiment, the presence of UR in the drinking water of pregnant mice reduced PALA‐induced embryolethality in day 10, but not day 8, embryos. These results indicate that the embryotoxic effects of PALA are the result of ATCase inhibition; furthermore, they suggest that the relative insensitivity of the day 10 embryo to the lethal effects of PALA may result either from a greater availability of UR to the day 10 (versus the day 8) embryo, or from an enhanced ability of the day 10 placenta to bind PALA and prevent its passage into the embryo.
The development of resistance to initially successful cancer therapies is a major cause of the morbidity and mortality associated with cancer. Identifying evolving resistance at an early stage could inform clinical decision making to adapt therapies before resistant cancer cell phenotypes have become clonally dominant or metastasized. This goal of early detection has prompted heavy investments in liquid biopsy, organoid, and high-throughput screening methodologies. Recently, High-Speed Live-Cell Interferometry (HSLCI), a quantitative phase imaging (QPI) methodology, was shown to predict triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) sensitivity to carboplatin only 40 hours after tumor removal from a mouse. Before HSLCI can be tested in the clinic, it must be adapted for minimally invasive sample acquisition techniques, throughput must be increased to enhance drug screening capacity, and, like any screening method, protocols must be adjusted to reflect drug-specific effects. To overcome these barriers, the system's hardware was redesigned to increase throughput six-fold and enable the simultaneous screening of multiple therapeutics, and experiments were expanded to include several new classes of drugs. Additionally, the updated system was then incorporated into fine needle biopsy-compatible protocols that were developed for HSLCI and yielded data concordant with recently-published in vivo screens.
We report the development of high-speed live-cell interferometry (HSLCI), a new multisample, multidrug testing platform for directly measuring tumor therapy response via real-time optical cell biomass measurements. As a proof of concept, we show that HSLCI rapidly profiles changes in biomass in BRAF inhibitor (BRAFi)-sensitive parental melanoma cell lines and in their isogenic BRAFi-resistant sublines. We show reproducible results from two different HSLCI platforms at two institutions that generate biomass kinetic signatures capable of discriminating between BRAFi-sensitive and -resistant melanoma cells within 24 h. Like other quantitative phase imaging (QPI) modalities, HSLCI is well-suited to noninvasive measurements of single cells and cell clusters, requiring no fluorescence or dye labeling. HSLCI is substantially faster and more sensitive than field-standard growth inhibition assays, and in terms of the number of cells measured simultaneously, the number of drugs tested in parallel, and temporal measurement range, it exceeds the state of the art by more than 10-fold. The accuracy and speed of HSLCI in profiling tumor cell heterogeneity and therapy resistance are promising features of potential tools to guide patient therapeutic selections.
Cell size and growth are tightly regulated processes balancing synthesis and metabolism to ensure proper cell function. Deviations from normal growth in response to drug treatment provide insights into the induced cellular dysfunction caused by pharmacotherapy. These changes in biomass growth have shown promise as a marker for drug sensitivity. However, like many biomarkers, the output cannot be treated as binary. Just as cancer cells are heterogenous on the molecular level, individual cells' biomass response to drug treatment can range from cell death to no effect. It is therefore important to begin to survey for the full range of biomass growth responses to drug treatment to understand the dysfunction induced. Here, we explore the response of different cancer cell lines to treatments that induce biomass growth changes ranging from apoptosis to senescence to simply delayed regrowth. These longer-term studies, ranging up to six days of constant monitoring, aid in the interpretation of more commonly performed shorter-term biomass growth experiments and identify cells of interest for further molecular characterization in these cell lines.
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Patients undergoing stem cell transplantation (SCT) are at risk for graft-versus-host disease (GVHD). It is not yet possible to discern in real-time which patients are at risk for GVHD as post-transplant immunosuppression is weaned. In this pilot study, we examined the utility of single-cell, T cell mass measurements as a biomarker for GVHD risk. Based on prior work demonstrating significant mass differences between activated and resting T cells, our group studied a model in which donor T cells exhibit a measurable increase in mass upon activation from exposure to recipient antigens. HLA-matched allogeneic (n = 14) and autologous SCT (n = 4) patients were enrolled. Myeloablative allogeneic SCT was performed with either HLA-matched unrelated (n = 11) or related (n = 3) donors. Samples (3 mL) were obtained from donor products and from the recipients (whole blood) at days 14, 30, 60 and 100 post-transplant. CD3+ cells were immunomagnetically isolated from each sample and analyzed (mean number of cells imaged per time point: nProduct = 1550, nD14 = 400, nD30 = 657, n60 = 605, nD100 = 709) by Live-Cell Interferometry (LCI), a novel, high-precision (<1% coefficient of variation) microscopy technique [LCI uses a specialized camera to measure local phase shifts, enabling the automated and non-invasive measurement of the masses of thousands of living cells with picogram (pg) sensitivity (Figure 1, Figure 2)].Figure 23D rendering of T cells from an LCI image.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Of the patients that underwent allogeneic SCT, 9 (69%) developed GVHD. Median onset of acute GVHD (n = 7) and chronic GVHD (n = 4) were at 58 and 126 days post-transplant, respectively. The mean mass of T cells from infused stem cell products was 68 pg for autologous SCT recipients and 58 pg for allogeneic SCT recipients. Post-transplant variability in T cell masses was observed in allogeneic SCT recipients. Patients who did not develop GVHD exhibited an initial (day 30) departure from, and later (day 60) restoration of T cell mass to levels similar to the infused stem cell product. Autologous SCT patients displayed similar kinetics. Together, autologous and allogeneic SCT patients free of GVHD exhibited no significant difference between infused product and day 100 normalized median T cell masses (p = .30). Conversely, patients with GVHD exhibited a significant (p = .006) difference between product and day 100 normalized median masses, showing a persistent elevation (~18% higher median T cell masses than infused stem cell product) (Figure 3). Of the 4 patients with a 20% or greater increase in mass at day 100, 2 developed grade 3-4 acute GVHD and one, severe chronic GVHD. LCI is an inexpensive and simple technique which provides a clinically-applicable assay that may help predict the risk of alloreactivity (GVHD) by measuring the departure from infused T cell mass. These data may be used to guide withdrawal of immunosuppression following SCT, and reduce the likelihood of GVHD or relapse occurring.
Patients undergoing high dose therapy and stem cell transplantation (SCT) develop mucosal toxicity with declining oral intake and nutrient absorption. Vitamin C (VitC) is critical in regulating the inflammatory response. Low vitamin C levels have been associated with poor outcomes in patients with systemic inflammatory state, such as is commonly observed in patients undergoing myeloablative conditioning and SCT. This study was performed to determine plasma VitC steady state levels in these patients. In this single institution IRB-approved prospective study, patients were enrolled prior to SCT and plasma VitC levels were measured at baseline and on days 0, 14, 30, and 60 post-transplant. Thus far, 15 patients have been studied, 4 underwent an autologous and 11 an allogeneic SCT (3 HLA matched related donors and 8 unrelated donors): median age was 50 years (range 31, 65) at the time of transplant. Primary diagnoses included myelofibrosis (2), AML (2), ALL (2), MDS (3) non-Hodgkin lymphoma (3), Hodgkin's lymphoma (1), Myeloma (1) and CML (1). Preparatory regimens were all myeloablative; Flu/Mel/ATG (2), Bu/Cy/ATG (5), BEAM (3), Bu/Flu/ATG (2), TBI/Cy/ATG (1), Mel 200 (1), Bu/Flu (1). Mucositis was observed in 12 patients, grades 1 (3), 2 (5) and 3 (4) with grades 2 and 3 possibly associated with Busulfan in the conditioning regimen. GVHD prophylaxis included tacrolimus with Mycophenolate (3) or Methotrexate (5) or cyclosporine with Mycophenolate (1) or Methotrxate (2). (Mean plasma VitC levels was 40.8 µmol/l (±18.4) (normal range: 50-80 µmol/L) at baseline, before falling to 27.3 µmol/l (±14.1) at day 0 (P < .05, c/w baseline) and reaching nadir at 21.5 µmol/l (±13.8) on day 14 (P < .05 c/w baseline) post-SCT (Figure 1). Plasma VitC levels recovered to 34.2 µmol/l (±20.5) at day 30, and subsequently to 37.2 µmol/l (±27.9) at day 60 (P < .05 c/w day 14). Of the patients studied, none maintained normal VitC levels throughout the study period and 13/15 developed significantly deficient VitC levels of <28 µmol/l.Patients with more severe WHO Grade 2-3 mucositis tended to have lower VitC levels at day 14 (Figure 2). VitC deficiency is an increasingly recognized factor in development of infection and poor outcomes in sepsis. In this study, transplant patients were found to have deficient plasma VitC levels following myeloablative conditioning. Parenteral VitC replacement therapy during the first two weeks following conditioning may alleviate mucositis, modulate inflammatory responses and reduce endothelial injury as well as allo-immune complications, i.e. GVHD following myeloablative conditioning therapy.Figure 2Relationship between Vitamin C levels and mucositis at Day 14.View Large Image Figure ViewerDownload Hi-res image Download (PPT)
Abstract The clinical outcome of allogeneic hematopoietic stem cell transplantation (SCT) is strongly influenced from the complications arising during the post-transplant immune restoration and has been well studied and described. However, the metabolic status of the recipient pre-transplant also has the potential to influence this outcome and has never been studied before and has the potential to enable risk stratification with respect to the development of transplant associated complications such as graft vs. host disease (GVHD). In order to better understand this aspect of transplant related complications we investigated the pre-transplantation metabolic signature to assess the possibility of pre-transplant risk stratification. This pilot study was composed of 14 patients undergoing myeloablative conditioning followed by either HLA matched related, unrelated donor, or autologous stem cell transplantation. Blood samples were taken prior to transplant and the plasma was comprehensively characterized with respect to its lipidome and metabolome via LCMS and GCMS. The results indicated a significantly pro-inflammatory metabolic profile in patients who eventually developed Graft vs. Host Disease (GVHD). The data revealed 5 potential pre-transplant biomarkers (1-monopalmitin, diacylglycerol (DG) 38:5, DG 38:6, 2-aminobutyric acid, and fatty acid (FA) 20:1) that demonstrated high sensitivity and specificity towards predicting post-transplant GVHD development. The predictive model developed demonstrated an estimated predictive accuracy of risk stratification of 100%, with an Area under the Curve of the ROC of 0.995 with 100%. The likelihood ratio of 1-monopalmitin (infinity), DG 38:5 (6.0) and DG 38:6 (6.0) also demonstrated that a patient with a positive test result for these biomarkers pre-transplant will likely have very high odds of developing GVHD post-transplant. Collectively the data demonstrates the possibility of using pre-transplant metabolic signature for risk stratification of SCT recipients with respect to development of GVHD.
