The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec‐1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild‐type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and “rosettes” (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near‐complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.
Mounting clinical and preclinical evidence demonstrates an important role for the intestinal microbiome in mediating efficacy of immune checkpoint inhibitor (ICI) antibody therapy across a number of tumor contexts. We sought to determine the effects of microbiome modulation on ICI therapy in a clinically recapitulative orthotopic murine lung cancer model. The microbiome of C57Bl/6NHsd mice was sterilized with antibiotic (ampicillin, streptomycin and colistin) for ten days, resulting in a 99.9% mean decrease in fecal aerobic and anaerobic bacterial load in comparison to naïve mice, handled under specific pathogen free (SPF) conditions. Luciferase-expressing murine Lewis lung carcinoma cells (LL/2-Red-FLuc) were surgically implanted into the left lung parenchyma of all animals. Animals were treated with locoregional radiotherapy (2x 9Gy fractions) targeted to the left lung. The gastrointestinal microbiome was reconstituted via oral gavage Q3D of ~1e09/dose commensal A. muciniphila (A. muc) and E. hirae (E. hir) over five doses or sterile saline as control and animals were randomized within commensal/saline treatment groups to equivalent mean tumor burden as measured by Lumina Series III In-Vivo Imaging System (IVIS). Animals were then administered anti-murine PD-1 or isotype control (0.25 mg) antibody (Ab) treatments Q3D over four doses via intraperitoneal injection. Tumor growth was monitored by IVIS over the course of the study, and ex-vivo IVIS was performed on lungs at endpoint (Day 12 post-tumor implantation). Tumor growth of microbiota non-reconstituted antibiotic-sterilized animals was only slightly inhibited by anti-PD-1 therapy. Animals administered commensal A. muc and E. hir and treatments also displayed slightly inhibited tumor growth kinetics, similar to those observed under saline/anti-PD-1 therapy. Fecal microbial sequencing and immunophenotypic analyses are ongoing. This study demonstrates the utility and ongoing development of a clinically recapitulative contextually accurate preclinical murine lung cancer model to assess the effects of specific microbiota in mediating the efficacy of anti-tumor immunotherapy. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Nicholas Rivelli, Alexandra Kury, Sallyann Vu, Gavin Gagnon, Veronica Ritchie, Kasey Reardon, Catarina Costa, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1499.
Humanized immune system (HIS) mice - immunocompromised Nod.Cg-Prkdcscid Il2rgtm1Wjl/SzJ animals in which the immune system has been humanized by the engraftment of CD34+ hematopoietic stem cells (HSC), peripheral blood mononuclear cells (PBMC), or other human cells/tissues - represent an important tool in pre-clinical oncology research. Here we investigated several pre-conditioning regimens (Busulfan pre-treatment or total body irradiation (TBI)) to determine which would yield maximum human leukocyte engraftment and lineage diversity. Animals were pre-treated either one or two doses of Busulfan (25 mg/kg) or varying doses of TBI (1.75, 2.0, or 2.5 Gy). One to two days later, animals received an adoptive transfer of native hCD34+ HSCs. Engraftment was monitored by flow cytometry assessment of peripheral blood four weeks after adoptive-transfer, and every four weeks thereafter through experiment termination at Week 16. Upon termination, spleen and bone-marrow were assessed for various leukocyte populations by flow cytometry. Animals undergoing pre-conditioning displayed increased human CD45+ cell engraftment at all timepoints as compared to animals without pre-conditioning, with animals treated with two doses of Busulfan demonstrating the greatest human CD45+ engraftment at all timepoints and reaching statistical significance at endpoint (vs no pre-conditioning; p<0.05). Diversity of leukocyte cell types (B cells, T cells, NK cells, and other hCD45+) in the bone-marrow was similar across pre-treatment paradigms, however for some lineages, representation in the bone-marrow was significantly altered between pre-conditioning type. The absolute number of T or B cells were increased in TBI and Busulfan treated groups, respectively, whereas NK cells as a percentage of singlets were increased in TBI treated groups. The diversity of leukocyte cell types in the spleen was likewise similar across groups, with B cells dominating. These data provide important insights into the utility of various pre-conditioning regimens for the humanization of NSG mice for pre-clinical oncology research, and demonstrate that the optimum pre-conditioning regimens should be selected based on treatment target. Citation Format: Caitlin S. Parello, Benjamin G. Cuiffo, Alexandria Kury, Kasey Reardon, Brett Van Dam, Sallyann Vu, Catarina Costa, Veronica Ritchie, Gavin Gagnon, Timothy Bateson, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. An assessment of pre-conditioning regimens for optimal hCD34+ hematopoietic stem cell humanization of NSGTMmice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 511.
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